Faculty Bibliography

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

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

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.

BACKGROUND: Low RNA yields from small tissue samples can limit the use of oligonucleotide microarrays (Affymetrix GeneChips). Methods using less cRNA for hybridization or amplifying the cRNA have been reported to reduce the number of transcripts detected, but the effect on realistic experiments designed to detect biological differences has not been analyzed. We systematically explore the effects of using different starting amounts of RNA on the ability to detect differential gene expression. RESULTS: The standard Affymetrix protocol can be used starting with only 2 micrograms of total RNA, with results equivalent to the recommended 10 micrograms. Biological variability is much greater than the technical variability introduced by this change. A simple amplification protocol described here can be used for samples as small as 0.1 micrograms of total RNA. This amplification protocol allows detection of a substantial fraction of the significant differences found using the standard protocol, despite an increase in variability and the 5' truncation of the transcripts, which prevents detection of a subset of genes. CONCLUSIONS: Biological differences in a typical experiment are much greater than differences resulting from technical manipulations in labeling and hybridization. The standard protocol works well with 2 micrograms of RNA, and with minor modifications could allow the use of samples as small as 1 micrograms. For smaller amounts of starting material, down to 0.1 micrograms RNA, differential gene expression can still be detected using the single cycle amplification protocol. Comparisons of groups of four arrays detect many more significant differences than comparisons of three arrays

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

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

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

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

Integrative optical imaging was used to show that long-chain synthetic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) polymers in a range of molecular weights from 7.8 to 1057 kDa were able to diffuse through the extracellular space in rat neocortical slices. Tortuosity (square root of ratio of diffusion coefficient in aqueous medium to that in brain) measured with such polymers averaged 1.57, a value similar to that obtained previously with tetramethylammonium, a small cation. When PHPMA was conjugated with bovine serum albumin (BSA) to make a bulky polymer with molecular weight 176 kDa, the tortuosity rose to 2.27, a value similar to that obtained previously with BSA alone and with 70-kDa dextran. The method of image analysis was justified with diffusion models involving spherical and nonspherical initial distributions of the molecules

Diffusion of molecules in brain extracellular space is constrained by two macroscopic parameters, tortuosity factor lambda and volume fraction alpha. Recent studies in brain slices show that when osmolarity is reduced, lambda increases while alpha decreases. In contrast, with increased osmolarity, alpha increases, but lambda attains a plateau. Using homogenization theory and a variety of lattice models, we found that the plateau behavior of lambda can be explained if the shape of brain cells changes nonuniformly during the shrinking or swelling induced by osmotic challenge. The nonuniform cellular shrinkage creates residual extracellular space that temporarily traps diffusing molecules, thus impeding the macroscopic diffusion. The paper also discusses the definition of tortuosity and its independence of the measurement frame of reference