Biomedical subjects

Charles Nicholson

Publications and source records attributed to Charles Nicholson.

13 recordsLinked to original sources

PubMed2006

In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space.

Diffusion within the extracellular space (ECS) of the brain is necessary for chemical signaling and for neurons and glia to access nutrients and therapeutics; however, the width of the ECS in living tissue remains unknown. We used integrative optical imaging to show that dextrans and water-soluble quantum dots with Stokes-Einstein diameters as large as 35 nm diffuse within the ECS of adult rat neocortex in vivo. Modeling the ECS as fluid-filled "pores" predicts a normal width of 38-64 nm, at least 2-fold greater than estimates from EM of fixed tissue. ECS width falls below 10 nm after terminal ischemia, a likely explanation for the small ECS visualized in electron micrographs. Our results will improve modeling of neurotransmitter spread after spillover and ectopic release and establish size limits for diffusion of drug delivery vectors such as viruses, liposomes, and nanoparticles in brain ECS.

Animals↗

PubMed2005

Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents.

Spreading depression (SD) involves current flow through principal neurons, but the pattern of current flow over the expanse of susceptible tissues or individual principal neurons remains undefined. Accordingly, tissue and single cell maps made from digital imaging of voltage-sensitive dye changes in hippocampal organotypic cultures undergoing SD were processed via optical current source density analysis to reveal the currents associated with pyramidal neurons. Two distinctive current flow patterns were seen. The first was a trilaminar pattern (420 microm2) that developed with the onset of SD in CA3 pyramidal neurons, in which SD most often began. This initial pattern comprised a somatic current sink with current sources to either side in the dendrites that lasted for seconds extending into the first aspect of the classical "inverted saddle" interstitial direct current waveform of SD. Next, the somatic sink backpropagated at a speed of millimeters per minute into the proximal dendrites, resulting in a reversal of the initial current flow pattern to its second orientation, namely dendritic sinks associated with a somatic source. The latter persisted for the remainder of SD in CA3 and was the only pattern seen in CA1, in which SD was rarely initiated. This backpropagating SD current flow resembles that of activity-dependent synaptic activation. Retrograde and associative signaling via principal neuron current flow is a key means to affect tissue function, including synaptic activation and, by extension, perhaps SD. Such current-related postsynaptic signaling might not only help explain SD but also neuroprotection and migraine, two phenomena increasingly recognized as being related to SD.

Animals↗

PubMed2005

Drug utilization in the pediatric intensive care unit: monitoring prescribing trends and establishing prioritization of pharmacotherapeutic evaluation of critically ill children.

The primary objective of this study was to characterize the drug exposure for children hospitalized in the authors' institution's pediatric intensive care unit for the year 2002. Secondary objectives included the examination of drug utilization differences among various age criteria and the suitability of the most prevalent resources for pediatric dosing guidance. Many of the most commonly prescribed agents in the pediatric intensive care unit fall into the broad categories of pain management/sedation and anti-infectives. Based on the generally narrow windows afforded by each of these drug classes, it is obvious that more, well-defined investigations in critically ill children are warranted. The existing dosing guidance for many of these agents is neither generalizable nor sufficient to accommodate the diversity in pediatric intensive care unit patients, and the current drug monographs fall short of any practical dosing information.

Adolescent↗

PubMed2004

Diffusion of epidermal growth factor in rat brain extracellular space measured by integrative optical imaging.

Epidermal growth factor (EGF) stimulates proliferation, process outgrowth, and survival in the CNS. Understanding the actions of EGF necessitates characterizing its distribution in brain tissue following drug delivery or release from cellular sources. We used the integrative optical imaging (IOI) method to measure diffusion of fluorescently labeled EGF (6,600 Mr; 4 microg/ml) in the presence of excess unlabeled EGF (90 microg/ml) to compete off specific receptor binding and reveal the "true" EGF diffusion coefficient following injection in rat brain slices (400 microm). The effective diffusion coefficient was 5.18 +/- 0.16 x 10(-7) (SE) cm2/s (n = 22) in rat somatosensory cortex and the free diffusion coefficient, determined in dilute agarose gel, was 16.6 +/- 0.12 x 10(-7) cm2/s (n = 27). Tortuosity (lambda), a parameter representing the hindrance imposed on EGF by the convoluted brain extracellular space (ECS), was 1.8, the lowest yet measured by IOI for a protein in brain. Control experiments with fluorescent dextran of similar molecular weight and tetramethylammonium confirmed EGF did not affect local ECS structure. We conclude that transport of smaller growth factors such as EGF through brain ECS is less hindered than that of larger proteins (>10,000 Mr, e.g., nerve growth factor) where typically lambda > 2.1. Modeling was used to predict that low lambda will allow EGF sources in the brain to be further from target cells and still elicit a biological response. High lambda values for larger growth factors imply more constrained local biological effects than with smaller proteins such as EGF.

Amino Acid Sequence↗

PubMed2004

Contribution of dead-space microdomains to tortuosity of brain extracellular space.

The extracellular space (ECS) of the brain is a major channel for intercellular communication, nutrient and metabolite trafficking, and drug delivery. The dominant transport mechanism is diffusion, which is governed by two structural parameters, tortuosity and volume fraction. Tortuosity (lambda) represents the hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium, while volume fraction (alpha) is the proportion of tissue volume occupied by the ECS. Diffusion of small ECS markers can be exploited to measure lambda and alpha. In healthy brain tissue, lambda is about 1.6 but increases to 1.9-2.0 in pathologies that involve cellular swelling. Previously it was thought that lambda could be explained by the circumnavigation of diffusing molecules around cells. Numerical models of assemblies of convex cells, however, give an upper limit of about 1.23 for lambda. Therefore, additional factors must be responsible for lambda in brain. In principle, two mechanisms could account for the measured value: a more complex ECS geometry or an extracellular macromolecular matrix. Here we review recent work in ischemic tissue suggesting concave geometrical formations, dead-space microdomains, as a major determinant of extracellular tortuosity. A theoretical model of lambda based on diffusion dwell times supports this hypothesis and predicts that, in ischemia, dead spaces occupy approximately 60% of ECS volume fraction leaving only approximately 40% for well-connected channels. It is further proposed that dead spaces are present in healthy brain tissue where they constitute about 40% of alpha. The presence of dead-space microdomains in the ECS implies microscopic heterogeneity of extracellular channels with fundamental implications for molecular transport in brain.

Animals↗

PubMed2003

Dead-space microdomains hinder extracellular diffusion in rat neocortex during ischemia.

During ischemia, the transport of molecules in the extracellular space (ECS) is obstructed in comparison with healthy brain tissue, but the cause is unknown. Extracellular tortuosity (lambda), normally 1.6, increases to 1.9 in ischemic thick brain slices (1000 microm), but drops to 1.5 when 70,000 Mr dextran (dex70) is added to the tissue as a background macromolecule. We hypothesized that the ischemic increase in lambda arises from diffusion delays in newly formed dead-space microdomains of the ECS. Accordingly, lambda decreases when dead-space diffusion is eliminated by trapping dex70 in these microdomains. We tested our hypothesis by analyzing the diffusion of several molecules in neocortical slices. First we showed that diffusion of fluorescent dex70 in thick slices declined over time, indicating the entrapment of background macromolecules. Next, we measured diffusion of tetramethylammonium (TMA+) (74 Mr) to show that the reduction of lambda depended on the size of the background macromolecule. The synthetic polymer, 40,000 Mr polyvinylpyrrolidone, reduced lambda in thick slices, whereas 10,000 Mr dextran did not. The dex70 was also effective in normoxic slices (400 microm) after hypoosmotic stress altered the ECS to mimic ischemia. Finally, the dex70 effect was confirmed independently of TMA+ using fluorescent 3000 Mr dextran as a diffusion marker in thick slices: lambda decreased from 3.29 to 2.44. Taken together, these data support our hypothesis and offer a novel explanation for the origin of the large lambda observed in ischemic brain. A semiquantitative model of dead-space diffusion corroborates this new interpretation of lambda.

Animals↗

PubMed2002

Measurement of diffusion parameters using a sinusoidal iontophoretic source in rat cortex.

A new method was developed to extract diffusion parameters in brain tissue using a sinusoidal iontophoretic point source of tetramethylammonium operated at different frequencies. The resulting steady state oscillating extracellular concentration of this probe molecule was continuously monitored using an ion-selective microelectrode located about 100 microm from the source. Because the probe molecules must diffuse through the extracellular space (ECS), the oscillating concentration at the recording location will develop a phase lag and an amplitude attenuation relative to the sinusoidal source. These two components of the signal can be analyzed to determine the tortuosity factor lambda and the ECS volume fraction alpha. The method also measures the nonspecific clearance rate constant kappa. In brain slices this reflects washout of diffusing molecules. Values of alpha (0.18+/-0.05) and lambda (1.67+/-0.08) obtained from this frequency method in rat cortical slices were similar to those obtained by the real-time iontophoretic method employing a square pulse source. The relative merits of the frequency method compared to the pulse method are discussed.

Animals↗

PubMed2002

Light scattering in rat neocortical slices differs during spreading depression and ischemia.

Spreading depression (SD) and ischemia are different pathophysiological events but have similar characteristics. This study investigated whether similarity exists in the light scattering (LS) properties during SD and ischemia in rat neocortical slices. SD was induced by injection of K(+) while ischemia was simulated by removing oxygen and glucose. LS was simultaneously recorded with changes in extracellular direct current (DC) potential and extracellular space (ECS) volume. LS was measured using a photon counting fiber optic system and the ECS volume change was determined by measuring the ECS concentration of tetramethylammonium (TMA(+)). Slices maintained in normal artificial cerebrospinal fluid (ACSF) showed a consistent LS increase during SD, but exhibited two different LS behaviors during 6 min of ischemia. In eight slices, LS decreased and remained so until the end of the ischemic challenge. In another 10 slices, LS diminished initially but, after 2 min, suddenly reversed sign, accompanied by a rapid negative shift in extracellular DC potential. When 50 or 91% of Cl(-) in the ACSF was replaced by membrane-permeable propionate, LS retained its increase during SD, but always showed the sudden LS reversal during ischemia. In contrast, when Cl(-) was substituted with membrane-impermeant methylsulfate, the SD-induced LS increase was replaced by an LS decrease, and the sudden LS reversal during ischemia was absent. While the LS signal showed different characteristics during SD and ischemia, the DC potential always presented negative shifts and the ECS volume always exhibited similar decreases. These results suggest that the polarity of the LS signal is determined by the competition of at least two factors: cell swelling and anion influx.

Action Potentials↗

PubMed2002

Independence of extracellular tortuosity and volume fraction during osmotic challenge in rat neocortex.

The structural properties of brain extracellular space (ECS) are summarised by the tortuosity (lambda) and the volume fraction (alpha). To determine if these two parameters were independent, we varied the size of the ECS by changing the NaCl content to alter osmolality of bathing media for rat cortical slices. Values of lambda and alpha were extracted from diffusion measurements using the real-time ionophoretic method with tetramethylammonium (TMA+). In normal medium (305 mosmol kg(-1)), the average value of lambda was 1.69 and of alpha was 0.24. Reducing osmolality to 150 mosmol kg(-1), increased lambda to 1.86 and decreased alpha to 0.12. Increasing osmolality to 350 mosmol kg(-1), reduced lambda to about 1.67 where it remained unchanged even when osmolality increased further to 500 mosmol kg(-1). In contrast, alpha increased steadily to 0.42 as osmolality increased. Comparison with previously published experiments employing 3000 M(r) dextran to measure lambda, showed the same behaviour as for TMA+, including the same constant lambda in hypertonic media but with a steeper slope in the hypotonic solutions. These data show that lambda and alpha behave differently as the ECS geometry varies. When alpha decreases, lambda increases but when alpha increases, lambda rapidly attains a constant value. A previous model allowing cellular shape to alter during osmotic challenge can account qualitatively for the plateau behaviour of lambda.

Animals↗

PubMed2002

Quantitative dual-probe microdialysis: evaluation of [3H]mannitol diffusion in agar and rat striatum.

Dual-probe microdialysis was used to study interstitial diffusion in the rat brain. A radiolabelled tracer, (3H]mannitol, was continuously infused at different concentrations via a probe acutely implanted into the striatum of an anaesthetized male rat or into a dilute agar gel. Samples were collected by a second probe placed 1 mm away from the first, and the recovered [3H]mannitol was measured by liquid scintillation counting. In the striatum, the delivery of [3H]mannitol was counteracted by its removal from the extracellular space by passive uptake into cells and clearance into the microcirculation, causing the diffusion profile to approach quasi steady-state levels within 2 h. Diffusion data from brain and agar were analysed using a mathematical model. The apparent (effective) diffusion coefficient for [3H]mannitol was D* = 2.9 x 10(-6) cm2/s, the effective volume fraction alpha* = 0.30 and the clearance rate constant kappa= 2.3 x 10(-5)/s. A tortuosity, lambda = 1.81, and penetration distance r = 4.2 mm, were calculated. We conclude that, using dual-probe microdialysis, parameters reflecting geometric and dynamic tissue properties may be obtained using appropriate mathematical analysis. Quantitative dual-probe microdialysis will be valuable in characterizing interstitial diffusion and the clearance processes underpinning volume transmission in the brain.

Agar↗

PubMed2002

Quantitative dual-probe microdialysis: mathematical model and analysis.

Steady-state microdialysis is a widely used technique to monitor the concentration changes and distributions of substances in tissues. To obtain more information about brain tissue properties from microdialysis, a dual-probe approach was applied to infuse and sample the radiotracer, [3H]mannitol, simultaneously both in agar gel and in the rat striatum. Because the molecules released by one probe and collected by the other must diffuse through the interstitial space, the concentration profile exhibits dynamic behavior that permits the assessment of the diffusion characteristics in the brain extracellular space and the clearance characteristics. In this paper a mathematical model for dual-probe microdialysis was developed to study brain interstitial diffusion and clearance processes. Theoretical expressions for the spatial distribution of the infused tracer in the brain extracellular space and the temporal concentration at the probe outlet were derived. A fitting program was developed using the simplex algorithm, which finds local minima of the standard deviations between experiments and theory by adjusting the relevant parameters. The theoretical curves accurately fitted the experimental data and generated realistic diffusion parameters, implying that the mathematical model is capable of predicting the interstitial diffusion behavior of [3H]mannitol and that it will be a valuable quantitative tool in dual-probe microdialysis.

Agar↗

PubMed2002

Theory relating in vitro and in vivo microdialysis with one or two probes.

In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungay et al. to provide a theoretical basis for in vitro and in vivo microdialysis. Specifically, we considered the effect of active clearance processes on in vivo microdialysis, and thereby elaborated the theory of Benveniste et al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on the in vitro and in vivo recoveries in dual-probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi-steady state is about 5.5 x 10(-5) s(-1). This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency.

Agar↗

PubMed2002

Water compartmentalization and spread of ischemic injury in thick-slice ischemia model.

Water compartmentalization was studied in a thick-slice (1000 microm) model of ischemia by combining water-content measurements with extracellular diffusion analysis. Thick slices bathed in artificial cerebrospinal fluid continually gained water. Total tissue water content was increased by 67% after 6 hours of the incubation. Diffusion measurements using the tetramethylammonium method showed that the extracellular space, typically occupying 20% of brain tissue in vivo, was decreased to 10% at 30 minutes and 15% at 6 hours in both deep and superficial layers of thick slices. Quantification of water compartmentalization revealed that water moved initially from the extracellular space into the cells. Later, however, both compartments gained water. The initial cell swelling was accompanied by dramatic shifts in potassium. An initial rise of extracellular potassium to about 50 mmol/L was measured with a potassium-selective microelectrode positioned in the center of the thick slice; the concentration decreased slowly afterwards. Potassium content analysis revealed a 63% loss of tissue potassium within two hours of the incubation. In thick slices, ionic shifts, water redistribution, and a loss of synaptic transmission occur in both deep and superficial layers, indicating the spread of ischemic conditions even to areas with an unrestricted supply of nutrients.

Animals↗