Danny O'Hare

QUANTITATIVE SPATIALLY RESOLVED MEASUREMENTS OF MASS TRANSFER THROUGH LARYNGEAL CARTILAGE

The scanning electrochemical microscope (SECM) is a scanned probe microscope that uses the response of a mobile ultramicroelectrode (UME) tip to determine the reactivity, topography, and mass transport characteristics of interfaces with high spatial resolution. SECM strategies for measuring the rates of solute diffusion and convection through samples of cartilage, using amperometric UMEs, are outlined. The methods are used to determine the diffusion coefficients of oxygen and ruthenium(III) hexamine [Ru(NH3)6(3+)] in laryngeal cartilage. The diffusion coefficient of oxygen in cartilage is found to be approximately 50% of that in aqueous electrolyte solution, assuming a partition coefficient of unity for oxygen between cartilage and aqueous solution. In contrast, diffusion of Ru(NH3)6(3+) within the cartilage sample cannot be detected on the SECM timescale, suggesting a diffusion coefficient at least two orders of magnitude lower than that in solution, given a measured partition coefficient for Ru(NH3)6(3+) between cartilage and aqueous solution, Kp = [Ru(NH3)6(3+)]cartilage/[RU(NH3)6(3+)]solution = 3.4 +/- 0.1. Rates of Ru(NH3)6(3+) osmotically driven convective transport across cartilage samples are imaged at high spatial resolution by monitoring the current response of a scanning UME, with an osmotic pressure of approximately 0.75 atm across the slice. A model is outlined that enables the current response to be related to the local flux. By determining the topography of the sample from the current response with no applied osmotic pressure, local transport rates can be correlated with topographical features of the sample surface, at much higher spatial resolution than has previously been achieved.

On the microelectrode behaviour of graphite–epoxy composite electrodes

Abstract Electrodes fashioned from conducting particles embedded in insulating binder show some properties commonly associated with arrays of microelectrodes, viz. independence of current from convective flow and sigmoidal current–voltage curves. We have systematically investigated the electrochemical behaviour of a range of composite formulations. The fraction of the surface area that is conducting and the size of the conducting features have been quantified using the novel technique of conducting atomic force microscopy (C-AFM). We have shown that flow sensitivity and voltammetric behaviour are correlated with results from the C-AFM. The more dilute formulations behave like arrays of microelectrodes, though exhibiting large time constants. The origin of this behaviour is discussed.

Electrochemical method for direct measurement of oxygen concentration and diffusivity in the intervertebral disc: electrochemical characterization and tissue-sensor interactions

A method was developed for the in vivo measurement of oxygen concentration and diffusivity in the intervertebral disc using a bare gold cathode. The effects of surface adsorption were quantified using electrochemical methods and it was demonstrated that valid data can be obtained despite interactions between the electrode and the macromolecules of the intervertebral disc. The technique is used to show the dependence of the oxygen transport properties on tissue water content.

Measurements of oxygenation and perfusion in skeletal muscle using multiple microelectrodes

This paper describes an apparatus to measure tissue oxygenation and perfusion (as measured by the wash-in rate of gaseous hydrogen) simultaneously at multiple points in muscle using needle microelectrodes. The development of suitable electrodes and apparatus is described, as well as the development of the method and its validation. In particular, the potential for tissue damage secondary to electrode insertion, the need for in vivo voltammetric determination of the operating potential and the extent of any electrode-tissue and of electrode--electrode interactions are explored, and are shown to be insufficient in magnitude to affect the technique. Its subsequent use to characterise oxygenation and perfusion in rabbit skeletal muscle at rest is also described. In resting tibialis anterior muscle of the rabbit the mean pO2 was 18 ± 13.3 mm Hg and the mean perfusion was 4.4 ± 1.3 ml s−1 100 g−1. There was a heterogeneity in simultaneously-measured values of pO2 and perfusion at different points within muscle, and also a temporal variation at the same site. The spans between the highest and lowest simultaneously-measured values of pO2 in muscle ranged from 14 to 80 mm Hg, and for perfusion, from 1 to 12 ml s−1 g−1. No significant correlation was evident from histological examination between either pO2 or perfusion and surrounding fibre type or capillary density

Cardiac hypertrophy impairs recovery from ischaemia because there is a reduced reactive hyperaemic response

OBJECTIVE The aim was to investigate whether impaired coronary flow reserve associated with cardiac hypertrophy could significantly limit the flow debt repayment following short periods of coronary occlusion and exacerbate or prolong episodes of myocardial ischaemia. METHODS Left ventricular hypertrophy was induced in guinea pigs by aortic constriction and the hearts were isolated six weeks later for Langendorff perfusion. Sham operated animals served as controls. The reactive hyperaemic response was studied following various lengths of occlusion of flow and the extent of aerobic and anaerobic metabolism was assessed in each group. RESULTS Heart weight/body weight ratio was increased by approximately 25% (P < 0.001) with aortic constriction. The reactive hyperaemic flow response in isolated hearts was impaired by hypertrophy in both magnitude (P < 0.05) and duration. The repayment of flow debt was also significantly reduced, suggesting an inadequate recovery of the myocardium following the occlusion. The total amount of O2 consumed by the heart throughout the duration of hyperaemia was less in hypertrophy than in sham operated controls, suggesting a decrease in aerobic metabolism. Total lactate discharge expressed as a ratio of O2 consumed, which provides an estimation of the degree of anaerobic in relation to aerobic metabolism, was greater in hypertrophy than in sham operated controls. CONCLUSIONS The hypertrophied heart is more vulnerable to brief periods of ischaemia because of an impaired reactive hyperaemic response which results in delayed metabolic recovery. These abnormalities may contribute to the increased morbidity associated with cardiac hypertrophy.

Myocardial tissue perfusion determined by particulate and diffusible tracers during ischaemia: what is measured?

OBJECTIVE Myocardial perfusion can be determined by many techniques which can be broadly divided into those employing particulate tracers and those employing diffusible tracers. The most commonly used particulate tracer is radioactive microspheres. However, as with other particulate tracers, they only determine convective transport from pre-capillary arterioles. If convective transport is the limiting factor in solute exchange, then particulate tracers will give comparable measurements to diffusible tracer techniques. However, if solute transport becomes diffusion-limited or alternative pathways of convective transport become more important, which may occur during regional ischaemia, perfusion visualised with clearance techniques using diffusible tracers may be greater than that determined with particulate tracers. This study set out to investigate this possibility in the rabbit myocardium under normal and ischaemic conditions. METHODS A pentobarbitone-anaesthetised rabbit model of regional ischaemia was used. Ischaemia of the apical region was induced by ligation of the large left ventricular branch of the circumflex artery. Tissue perfusion was determined by radioactive microspheres (n = 5) and the clearance of hydrogen, which was detected voltammetrically by platinum microelectrodes (n = 5). Measurements were made prior to and following coronary ligation and the ischaemic region was demarcated using the particulate tracer monastral blue. The exchange of diffusible solutes was visualised using digital fluorescence microscopy on histological sections of tissue following systemic administration of the fluorophore Evans blue labelled albumin (n = 4). RESULTS Coronary ligation produced an ischaemic zone occupying 50 +/- 13% of the left ventricle. In ischaemic tissue, flow determined by microspheres fell to 3.9 +/- 4.1% of its pre-ligation value, but solute exchange fell only to 22 +/- 10% (adjusted for changes in the partition coefficient of H2 during ischaemia, P < 0.05). Perfusion measured by microspheres and hydrogen clearance was unchanged in the non-ischaemic area during coronary ligation. There was preferential uptake of Evans blue albumin towards the endocardial surface in the ischaemic region and areas of local uptake through the ventricular wall, which were possibly associated with vessels. CONCLUSION This work demonstrates that under normal physiological conditions nutrient supply is determined by pre-capillary delivery. However, during ischaemia diffusive transport plays an increasingly important role. The alternative pathways for solute exchange are likely to have an important influence on the rate and extent of myocardial necrosis during coronary occlusion.

Investigation of the Endothelial Cell Glycocalyx using Electrophoresis

The electrophoretic mobilities of bovine and porcine endothelial cells were measured by means of a light-scattering technique. Cells were harvested mechanically and by collagenase or trypsin digestion, and measurements were made on both fresh and chemically fixed cells under a range of solution pH, ionic strength, and composition. The effects of adding serum albumin to the buffer solution were also investigated. Electrophoretic mobilities were comparable to those reported for cultured cells, and for bovine cells at pH 7.4 they ranged from -0.69 ±. 01 (μm/s)/ (V/cm) in 0.15 M NaCl to -2.04 ±. 08 (μn/s)/(V/cm) in 0.015 M NaCl. Cells harvested mechanically had the highest mobility and those released by trypsin the lowest. Mobility became positive at pH 3, indicating that the glycocalyx contains more basic than sulphate groups. Calcium reduced the mobility at acid and neutral pH and caused charge reversal at high pH. In the presence of bovine serum albumin (1% w/v), mobility was reduced, demonstrating charge ...

Oxygenation and perfusion of rabbit tibialis anterior muscle subjected to different patterns of electrical stimulation.

Dual amperometric microelectrodes were used to measure local pO2 and perfusion at multiple sites in the fast-twitch tibialis anterior muscles of anaesthetized rabbits. Six muscles were stimulated continuously at 10, 5, or 2.5 Hz. For all three frequencies, perfusion declined to about 50% of resting levels and recovered after stimulation. These changes corresponded to a rise followed by a fall in extracellular pO2. The highest levels of pO2 were reached during stimulation at 10 Hz. Eight muscles were stimulated tetanically at 100 Hz for 200 ms with duty cycles that were varied between 1.3 and 20.0%. Perfusion rose to 8.7 ± 2.0 ml s−1 100 g−1 at a duty cycle of 5% and declined with further increases in duty cycle. pO2 was depressed for duty cycles less than 10% but rose above resting levels at higher duty cycles. It is suggested that the paradoxical combination of elevated pO2 and depressed perfusion is attributable to stimulation conditions that exceed the oxygen transport capacity of a fast muscle.

A novel in vivo nitric oxide sensor

Graphite–epoxy electrodes modified with N,N′-o-phenylenebis(salicylidineiminato)iron(III) are shown to be capable to detecting nitric oxide in vivo.