Pernilla Abrahamsson

Optimised sample handling in association with use of the CMA 600 analyser.

A large degree of variability for batched analysis of serially collected microdialysis samples measured with the CMA 600 analyser has been described. This study was designed to identify sources of variability related to sample handling. Standard concentrations of four solutes were placed in microdialysis vials and then stored and analysed at intervals. Results were analysed for variability related to vial and cap type, duration and temperature of storage, centrifugation and re-analysis. The main results were that centrifugation of samples reduced variability. When a batch of 24 samples was analysed, the use of crimp caps reduced evaporation. Samples in glass vials with crimp caps could be stored in a refrigerator for up to 14 days without large variability in concentration compared to plastic vials which demonstrated variability already when stored for more than 1 day. We conclude that variability in microdialysis results can occur in relation to storage and analysis routines if routines are not optimised concerning evaporation. Centrifugation before analyses, glass vials with crimp caps even during frozen storage, and attention to minimal times for samples to be uncapped during analysis all contribute to minimise variability in the handling and analysis of microdialysis samples.

Detection of myocardial ischaemia using surface microdialysis on the beating heart

Microdialysis (MD) can be used to study metabolism of the beating heart. We investigated whether microdialysis results obtained from epicardial (surface) sampling reflect acute changes in the same way as myocardial sampling from within the substance of the ventricular wall. In anaesthetized open‐thorax pigs a coronary snare was placed. One microdialysis probe was placed with the sampling membrane intramyocardially (myocardial), and a second probe was placed with the sampling membrane epicardially (surface), both in the area which was made ischaemic. Ten minutes collection intervals were used for microdialysis samples. Samples from 19 pigs were analysed for lactate, glucose, pyruvate and glycerol during equilibration, baseline, ischaemia and reperfusion periods. For both probes (surface and myocardial), a total of 475 paired simultaneous samples were analysed. Results from analyses showed no differences in relative changes for glucose, lactate and glycerol during baseline, ischaemia and reperfusion. Surface microdialysis sampling is a new application of the microdialysis technique that shows promise and should be further studied.

Metabolic responses in ischemic myocardium after inhalation of carbon monoxide

Background: To clarify the mechanisms of carbon monoxide (CO) tissue‐protective effects, we studied energy metabolism in an animal model of acute coronary occlusion and pre‐treatment with CO.

Carbon monoxide concentration in donated blood: relation to cigarette smoking and other sources

BACKGROUND: Carbon monoxide (CO) is normally present in the human body due to endogenous production of CO. CO can also be inhaled by exposure to external sources such as cigarette smoke, car exhaust, and fire. The purpose of this study was to investigate CO concentrations in blood from 410 blood donors at the blood center in Umeå, Sweden. To further evaluate the effects of cigarette smoking on CO concentrations, the elimination time for CO was examined in six volunteer smokers after a smoked cigarette.

Does carbon monoxide treatment alter cytokine levels after endotoxin infusion in pigs? A randomized controlled study

BackgroundCarbon monoxide (CO) has recently been suggested to have anti-inflammatory properties, but data seem to be contradictory and species-specific. Thus, in studies on macrophages and mice, pretreatment with CO attenuated the inflammatory response after endotoxin exposure. On the other hand, human studies showed no effect of CO on the inflammatory response. Anti-inflammatory efficacy of CO has been shown at concentrations above 10% carboxyhaemoglobin. This study was undertaken to elucidate the possible anti-inflammatory effects of CO at lower CO concentrations.MethodsEffects of CO administration on cytokine (TNF-alpha, IL-6, IL-1beta and IL-10) release were investigated in a porcine model in which a systemic inflammatory response syndrome was induced by endotoxin infusion. Endotoxin was infused in 20 anaesthetized and normoventilated pigs. Ten animals were targeted with inhaled CO to maintain 5% COHb, and 10 animals were controls.ResultsIn the control group, mean pulmonary artery pressure increased from a baseline value of 17 mmHg (mean, n = 10) to 42 mmHg (mean, n = 10) following 1 hour of endotoxin infusion. Similar mean pulmonary artery pressure values were found in animals exposed to carbon monoxide. Plasma levels of all of the measured cytokines increased in response to the endotoxin infusion. The largest increase was observed in TNF-alpha, which peaked after 1.5 hours at 9398 pg/ml in the control group and at 13395 pg/ml in the carbon monoxide-exposed group. A similar peak was found for IL-10 while the IL-6 concentration was maximal after 2.5 hours. IL-1beta concentrations increased continuously during the experiment. There were no significant differences between carbon monoxide-exposed animals and controls in any of the measured cytokines.ConclusionOur conclusion is that 5% COHb does not modify the cytokine response following endotoxin infusion in pigs.

Ischaemic preconditioning reduces myocardial calcium overload in coronary-occluded pig hearts shown by continuous in vivo assessment using microdialysis

During ischaemia, ATP depletion leads to insufficient fuelling for Na+/K+ ATPase, decreased electrochemical potential and increased influx of calcium ions. This study demonstrated a means to assess the effects of ischaemic preconditioning (IP) on the free intracellular Ca2+ pool during prolonged ischaemia. In a porcine myocardial ischaemia model, microdialysis (MD) was used for sampling of metabolic and injury markers in IP and non‐IP (control) groups. 45Ca2+ was delivered in microperfusate locally to ischaemic myocardium, with distribution and uptake assessed by 45Ca2+ recovery in microdialysate. Cardiomyocytes in vitro were exposed to a Ca2+ ionophore and tested for 45Ca2+ uptake. An accentuated myocardial calcium ion influx (observed as an increased microdialysate 45Ca2+ recovery in the extracellular milieu) was noted in control pigs compared with IP pigs during ischaemia. Suspended cardiomyocytes preincubated with a Ca2+ ionophore to increase the intracellular calcium ion pool and subsequently incubated with 45Ca2+, displayed lower 45Ca2+ uptake in cells compared with control cells not exposed to the ionophore, corroborating the idea of a strong relationship between degree of intracellular calcium overload and microdialysate 45Ca2+ recovery. The ischaemic insult was differentially verified by metabolic and injury markers. We introduce an in vivo method for serial assessment of myocardial calcium overload during ischaemia, using a MD technique and 45Ca2+ inclusion. IP leads to relatively less calcium overload as assessed by this new method, and we interpret this to mean that reduction in calcium overload is an important part of the IP protective effect.

Surface microdialysis sampling: a new approach described in a liver ischaemia model.

We recently have shown that samples from microdialysis (MD) probes placed on the surface of the heart reflect metabolic events in the myocardium. This new interesting observation challenges us to consider whether surface application of MD applies to other parenchymatous organs and their surfaces. In 13 anesthetized pigs, transient liver ischaemia was achieved by occlusion of arterial and venous inflow to the liver. Two probes on liver surface and two in parenchyma were perfused with a flow rate of 1 μl per min (n = 13). An identical set‐up was used for probes with a flow rate of 2 μl per min (n = 9). Samples were collected for every 15‐min period during 60 min of baseline, 45 min of ischaemia and 60 min of reperfusion. Lactate, glucose, pyruvate and glycerol were analysed in MD samples. We focused on relative changes in the present study. There was a strong agreement in relative lactate and glucose levels between probes placed on liver surface and those on parenchyma. No significant differences in relative changes in lactate and glucose levels were seen between samples from surface probes and probes in liver parenchyma during equilibration, baseline, ischaemia or reperfusion with a flow rate of 1 μl per min. MD sampling applied on the liver surface is a new application area for the MD technique and may be used to monitor liver metabolism during both physiological and pathophysiological conditions.

Exogenous carbon monoxide does not affect cell membrane energy availability assessed by sarcolemmal calcium fluxes during myocardial ischaemia-reperfusion in the pig.

Carbon monoxide is thought to be cytoprotective and may hold therapeutic promise for mitigating ischaemic injury. The purpose of this study was to test low-dose carbon monoxide for protective effects in a porcine model of acute myocardial ischaemia and reperfusion. In acute open-thorax experiments in anaesthetised pigs, pretreatment with low-dose carbon monoxide (5% increase in carboxyhaemoglobin) was conducted for 120 min before localised ischaemia (45 min) and reperfusion (60 min) was performed using a coronary snare. Metabolic and injury markers were collected by microdialysis sampling in the ventricular wall. Recovery of radio-marked calcium delivered locally by microperfusate was measured to assess carbon monoxide treatment effects during ischaemia/reperfusion on the intracellular calcium pool. Coronary occlusion and ischaemia/reperfusion were analysed for 16 animals (eight in each group). Changes in glucose, lactate and pyruvate from the ischaemic area were observed during ischaemia and reperfusion interventions, though there was no difference between carbon monoxide-treated and control groups during ischaemia or reperfusion. Similar results were observed for glycerol and microdialysate 45Ca2+ recovery. These findings show that a relatively low and clinically relevant dose of carbon monoxide did not seem to provide acute protection as indicated by metabolic, energy-related and injury markers in a porcine myocardial ischaemia/reperfusion experimental model. We conclude that protective effects of carbon monoxide related to ischaemia/reperfusion either require higher doses of carbon monoxide or occur later after reperfusion than the immediate time frame studied here. More study is needed to characterise the mechanism and time frame of carbon monoxide-related cytoprotection.

Ischaemic pre-conditioning means an increased adenosine metabolism with decreased glycolytic flow in ischaemic pig myocardium

Background: Ischaemic pre‐conditioning (IP) is a potent protective mechanism for limiting the myocardial damage due to ischaemia. It is not fully known as to how IP protects. The metabolism of adenosine may be an important mechanistic component. We study the role of adenosine turnover together with glycolytic flow in ischaemic myocardium subjected to IP.

Outcome of microdialysis sampling on liver surface and parenchyma

BACKGROUND To investigate whether surface microdialysis (μD) sampling in probes covered by a plastic film, as compared to noncovered and to intraparenchymatous probes, would increase the techniques sensitivity for pathophysiologic events occurring in a liver ischemia-reperfusion model. Placement of μD probes in the parenchyma of an organ, as is conventionally done, may cause adverse effects, e.g., bleeding, possibly influencing outcome. METHODS A transient ischemia-reperfusion model of the liver was used in six anesthetized normoventilated pigs. μD probes were placed in the parenchyma and on the liver surface. Surface probes were either left uncovered or were covered by plastic film. RESULTS Lactate and glucose levels were significantly higher in plastic film covered probes than in uncovered surface probes throughout the ischemic period. Glycerol levels were significantly higher in plastic film covered probes than in uncovered surface probes at 30 and 45 min into ischemia. CONCLUSIONS Covering the μD probe increases the sensibility of the μD-technique in monitoring an ischemic insult and reperfusion in the liver. These findings confirm that the principle of surface μD works, possibly replacing need of intraparenchymatous placement of μD probes. Surface μD seemingly allows, noninvasively from an organs surface, via the extracellular compartment, assessment of intracellular metabolic events. The finding that covered surface μD probes allows detection of local metabolic changes earlier than do intraparenchymatous probes, merit further investigation focusing on μD probe design.