Jeffrey J. Kelly

Spatial Heterogeneity in Oligodendrocyte Lineage Maturation and Not Cerebral Blood Flow Predicts Fetal Ovine Periventricular White Matter Injury

Although periventricular white matter injury (PWMI) is the leading cause of chronic neurological disability and cerebral palsy in survivors of premature birth, the cellular-molecular mechanisms by which ischemia-reperfusion contributes to the pathogenesis of PWMI are not well defined. To define pathophysiologic relationships among ischemia, acute cerebral white matter damage, and vulnerable target populations, we used a global cerebral ischemia-reperfusion model in the instrumented 0.65 gestation fetal sheep. We developed a novel method to make repeated measurements of cerebral blood flow using fluorescently labeled microspheres to resolve the spatial heterogeneity of flow in situ in three-dimensional space. Basal flow in the periventricular white matter (PVWM) was significantly lower than in the cerebral cortex. During global cerebral ischemia induced by carotid occlusion, flow to all regions was reduced by nearly 90%. Ischemia of 30 or 37 min duration generated selective graded injury to frontal and parietal PVWM, two regions of predilection for human PWMI. Injury was proportional to the duration of ischemia and increased markedly with 45 min of ischemia to extensively damage cortical and subcortical gray matter. Surprisingly, the distribution of PVWM damage was not uniform and not explained by heterogeneity in the degree of white matter ischemia. Rather, the extent of white matter damage coincided with the presence of a susceptible population of late oligodendrocyte progenitors. These data support that although ischemia is necessary to generate PWMI, the presence of susceptible populations of oligodendrocyte progenitors underlies regional predilection to injury.

Cerebral blood flow heterogeneity in preterm sheep: lack of physiologic support for vascular boundary zones in fetal cerebral white matter

Periventricular white matter (PVWM) injury is the leading cause of neurologic disability in survivors of prematurity. To address the role of ischemia in PVWM and cerebral cortical injury, we hypothesized that immaturity of spatially distal vascular ‘end zones’ or ‘border zones’ predisposes PVWM to greater decreases in cerebral blood flow (CBF) than more proximal structures. We quantified regional CBF with fluorescently labeled microspheres in 0.65 gestation fetal sheep in histopathologically defined three-dimensional regions by post hoc digital dissection and coregistration algorithms. Basal flow in PVWM was significantly lower than in gyral white matter and cortex, but was equivalent in superficial, middle, and deep PVWM. Absolute and relative CBF (expressed as percentage of basal) did not differ significantly during ischemia or reperfusion between PVWM, gyral white matter, or cortex. Moreover, CBF during ischemia-reperfusion was equivalent in three adjacent PVWM levels and was not consistent with the magnitude of severity of PVWM injury, defined by TUNEL (terminal deoxynucleotidyltransferase-mediated dUPT nick end labeling) staining. However, the magnitude of ischemia was predicted by the severity of discrete cortical lesions. Hence, unlike cerebral cortex, unique CBF disturbances did not account for the distribution of PVWM injury. Previously defined cellular maturational factors, thus, appear to have a greater influence on PVWM vulnerability to ischemic injury than the presence of immature vascular boundary zones.

Regional blood flow measurements from fluorescent microsphere images using an Imaging CryoMicrotome

An automated image acquisition and analysis system has been developed that rapidly determines regional blood flow by using the locations of fluorescent microspheres deposited in tissue. A motor-driven microtome removes sections of frozen tissues in steps variable between 10 and 100 μm. Filtered light excites microsphere fluorescence from the exposed surface of the remaining tissue block. A charge coupled device camera records fluorescence images from the tissue block surface after removal of each slice. Approximately 450 images are analyzed from perfused rat hearts providing precise x, y, and z locations of about 10 000 microspheres. Image analysis of fluorescent microspheres is much faster and less labor intensive than traditional indirect microsphere-based flow measurements while providing higher quality data.

Imaging Epicardial Oxygen

AbstractMeasurements of oxygen concentration and metabolic status in the heart are important to understanding the mechanisms that control cardiac respiration and its response to changing workload and substrate delivery. This paper presents images, recorded from a perfused rat heart, that reveal regional changes in concentrations of epicardial oxygen and of mitochondrial NADH in response to local ischemia, heterogeneous perfusion, and barbiturate inhibition. A fluorescence/phosphorescence imaging system was developed to acquire digital images of oxygen concentration and NADH fluorescence from the epicardium of perfused hearts. The oxygen imaging technique is based upon quenching of Pd(II) meso-tetra(p-sulfonatophenyl)porphin phosphorescence by dissolved oxygen. Images of oxygen and NADH fluorescence provided complimentary information about oxygen supply and demand in the heart. The utility of two-dimensional measurements of the mitochondrial bioenergetic status is illustrated by the comparison of gradients for NADH and oxygen across the boundary separating locally ischemic tissue from normoxic epicardium. Images of oxygen concentration provide a powerful means for studying the dynamics of regional oxygen supply/demand relationships in cardiac muscle.

Tissue temperature by near-infrared spectroscopy

Optical determinations of tissue temperature by near-infrared (NIR) spectroscopy provides the basis for measuring localized changes in tissue metabolism associated with congnition, mechanical work, inflammation, or malignancy. Absorbance changes in NIR spectra of tissue water are shown to correlate with tissue sample temperature. Digital tissue transmission spectra of samples 1-5 mm thick of bovine and avain muscles were obtained over temperatures ranging from 17 to 45C. Reflectance spectra were obtained from blocks of porcine muscle over the temperature range 14 to 46C. Multilinear regression analysis of the correlation between absorbance or reflectance and tissue temperature demonstrated that each O-H bond onvertone spectral region (960, 1200, 1450, and 1920 nm) has a high correlation with tissue temperature. Transmission results gave standard error of the estimates (SEE) and standard error of prediction (SEP) from cross-validation analysis of 0.02 to 0.12C for SEE and 0.04 to 0.12C for SEP. Reflectance results gave SEEs of 0.06 to 0.24C. Combinations of O-H vibrational modes of water give rise to NIR absorbance in solution and tissue. The spectra show a regular shift to shorter wavelength absorbance as temperature increases. Such shifts may be due to decreasing hydrogen-bonding with increasing temperature. These studies have established that the temperature dependent changes in water NIR spectra can be utilized to evaluate tissue temperature with precision and accuracy.

Videofluorometer for imaging tissue metabolism

A videofluorometer is described that directly acquires digital metabolic images of reduced nicotinamide adenine dinucleotide (NADH) fluorescence in tissue. NADH fluorescence provides an intrinsic indicator of the state of tissue mitochondrial oxidative metabolism. The device combines a computer‐controlled fluorescence excitation system with digital image acquisition to quantify tissue bioenergetics in both spatial and time domains. Localized ischemia following coronary artery ligation in a perfused rat heart (model for a coronary artery occlusion heart attack) is used as an example to demonstrate the capabilities of the system. This videofluorometer permits monitoring changes in physiological state of organs and tissue without interfering with tissue metabolism. The digital nature of the acquired image allows detailed analysis of physiological features and their time dependence.

Simultaneous Determination of Hemoglobin and Myoglobin Oxygen Binding Curves by Spectral Curve Fitting

A completely optical method is described for the simultaneous determination of hemoglobin and myoglobin oxygen saturation. Solution oxygen concentrations were computed from measurements of phosphorescence decay of a soluble palladium porphyrin using the Stern-Volmer quenching relationship. Visible absorption spectra were recorded of hemoglobin/myoglobin mixtures progressively deoxygenated by bacterial aerobic metabolism. Separate hemoglobin and myoglobin oxygenation curves were resolved from the spectra of solutions containing both proteins by curve fitting, with the use of singular value decomposition of the spectra vs. oxygen concentration matrix. The method yielded a P50 of 1.2 Torr for horse heart myoglobin at 24 ± 1°C, while the P50 for sheep hemoglobin was 23 Torr at the same temperature. These values, obtained from mixtures, compare favorably with literature values determined for hemoglobin and myoglobin separately.

Myoglobin Oxygen Binding Curves Determined by Phosphorescence Quenching of Palladium Porphyrin

A completely optical method was developed for evaluating myoglobin-oxygen saturation. Solution oxygen concentrations were computed from measurements of phosphorescence decay of a soluble palladium porphyrin according to the Stern-Volmer quenching relationship. Visible absorption spectra were recorded of myoglobin solutions progressively deoxygenated by bacterial aerobic metabolism. Myoglobin oxygenation curves were obtained from the spectra by two full-spectrum procedures, singular value decomposition (SVD) and curve fitting, and by the traditional dual-wavelength isosbestic method. The calculated P50 for horse heart myoglobin at 24 ± 1°C was 1.2 Torr by SVD, 1.2 Torr by curve fitting, and 1.4 Torr by dual-wavelength spectroscopy. These results compare favorably with a value of 1.1 Torr estimated from the work of Rossi-Fanelli and Antonini for human myoglobin. The Hill coefficient, theoretically 1.00 for myoglobin, was experimentally determined to be 0.96 by SVD and 0.95 by curve fitting of the spectral data matrix.

Metabolic Spectroscopy for Monitoring Electromagnetic Medical Techniques

Electromagnetic diagnostic and treatment devices exert direct and indirect effects on tissue metabolism. Electromagnetic devices produce spatially inhomogeneous deposition of energy in tissues and, therefore, affect tissue metabolism heterogeneously. Optical spectroscopy provides unique approaches to monitoring tissue metabolic status during medical treatment. Methods are being developed that provide both area and depth resolved metabolic measurements. This emerging technology provides high contrast images of tissue metabolism, perfusion and oxygenation for use in monitoring effects of electromagnetic radiation on tissues.

Reflectance Measurement of Heart Muscle Oxygenation

Energy production in heart muscle requires adequate nutrient supply and oxygen delivery. The bioenergetic processes of the mitochondria utilize reduced equivalents from nutrients in the form of reduced NADH and FADH2, and oxidizing equivalents from oxygen to produce proton gradients for the formation of ATP during oxidative phosphorylation. Oxygen supply to red mammalian muscle includes the involvement of two heme-containing transport/buffer proteins. Hemoglobin (Hb) in red blood cells binds and transports oxygen from lungs to tissue, while myoglobin (Mb) provides temporary oxygen storage and buffering within muscle cells. When both Hb and Mb are present, they combine to increase the apparent oxygen concentration in solution by about 50-fold. Mitochondrial cytochromes aa 3 b and c play important roles as electron carriers in the mitochondrial electron-transport chain.