Willard C. Whitehouse

Cortical oxygen consumption and NADH fluorescence during metrazol seizures in normotensive and hypotensive cats

Abstract The relationship between the oxidation of intramitochondrial NADH and increased oxygen utilization previously described in various preparations has not yet been directly studied in large areas of the cerebral cortex during generalized seizures. In the present study, changes in the NADH fluorescence of one or both cortical hemispheres were measured with a television fluorometer during Metrazol seizures in normotensive and hypotensive states. Fluorescein fluorescence was used as a reference signal. Combined with measurements of sagittal sinus blood flow and oxygen saturation, this technique demonstrated generalized ictal decreases in NADH fluorescence, the time integrals of which linearly related to the integrals of simultaneous increases in relative cortical O 2 consumption during seizures occurring at normal blood pressures. Seizures observed during systemic hypotension and probable cerebral hypoperfusion, however, were characterized by generalized increases in NADH fluorescence, the integrals of which were not related to the integrals of relatively delayed increases in cortical O 2 consumption. Similar anomalous NADH increases during seizures were also occasionally seen in certain cortical areas in normotensive animals, suggesting local ischemia. In addition. NADH fluorescence and cortical O 2 consumption were found to be affected by changes in blood pressure in the absence of ictal activity. These observations suggest that cortical NADH fluorescence is dependent both on metabolic demand and O 2 availability, and consequently may be validly utilized as an indicator of activated cortical oxidative metabolism during seizures only under conditions of adequate cortical oxygenation.

An Automated Method for the Measurement of Ventricular Volume

Several important indices of myocardial performance depend upon accurate and frequent measurement of ventricular volume. Studies employing such measurements have been limited because of the difficulty of manually measuring and calculating volumes frequently enough to obtain meaningful data. We, therefore, have developed an automated method for determination of ventricular volume in man. Left ventricular cineangiograms taken in the right anterior oblique position at 60 frames/sec are projected with a flickerless projector onto a Plumbicon television camera. A second television camera is used by a skilled operator for masking out noncontributory portions of the film and for shading selected areas to facilitate accurate recognition of the opacified chamber. An electronic video-tracking device then simultaneously determines the area and the maximum length of the opacified chamber in each cine frame. These data are recorded as analog signals on magnetic tape. Volumes are calculated by computer and plotted against time. When volumes determined by this automated method are compared with those obtained by manual planimetry, the correlation coefficient is 0.96. Aluminum prolate spheroids, left ventricular casts, and left ventricular cineangiograms were studied. This automated technic permits rapid and accurate measurement of ventricular volume in patients having diagnostic left ventriculograms.

Effects of Ischemia on the Removal of Extracellular Potassium in Cat Cortex during Pentylenététrazol Seizures

Summary: Changes in cortical extracellular potassium activity ([K+]o), NADH fluorescence, and oxygen consumption were studied in anesthetized cats during pentylenetetrazol seizures. The effects of partial ischemia induced by either hypotension or intermittent carotid artery occlusion on these parameters were investigated. Nonischemic seizures were characterized by gradual generalized decreases in cortical NADH fluorescence and increases in 02 consumption, along with rapid increases in [K+]0, which then usually fell slightly as the ictal discharge continued. Ischemic seizures, on the other hand, were accompanied by complex changes in NADH fluorescence, by smaller delayed maximal increases in O2 consumption that lasted beyond the end of ictal activity, and by more sustained increases in [K+]0. The decay of [K+]0 after the termination of seizures in both nonischemic and moderately ischemic animals was not a monoexponential function: plots of lnΔ[K+]0 versus time showed an initial linear decline (of slope M1) that rather abruptly slowed (to slope M2) after 2 to 5 sec and then often increased to the original rate. Both M, and M2 were proportionately decreased by ischemia. In addition, the rate of [K+]0 removal could be slowed by acute ischemia induced either during or after the end of ictal activity. The initial rate of postictal [K+]0 removal (M1) was found to be linearly and inversely related to the level of cortical NADH fluorescence at the time of seizure termination. The results of this study suggest that an O2‐dependent transport mechanism plays a major role in the removal of [K+]0 during and following the termination of generalized pentylenetetrazol seizures in the cat.

Sodium fluorescein: a new reference for NADH fluorometry

Fluorometry of the NADH (nicotinamide adenine dinucleotide) redox state in various tissues is performed by illuminating a given preparation with ultraviolet light (UV) and densitometrically measuring the specific fluorescence of N A D H (peak: 450 nm; range: 425-475 nm)l,~,5,6, 9. In order to compensate for the absorption and scattering of 450 nm light by blood, diffusely reflected UV is commonly used as a reference signal which is subtracted from the N A D H fluorescence during changes in the redox state. The frequent presence of specularly reflected UV, which changes unpredictably with tissue motion, limits N A D H fluorometry to either very small, carefully chosen tissue areas1, 6 or to relatively motionless preparations~, 7. Situations in which larger areas of tissue are to be studied, especially in the presence of significant motion such as might be encountered during large increases in blood flow, require an NADH reference which is optically affected in a way equal to N A D H fluorescence by blood flow changes, but is independent of specular reflectance changes. Sodium fluorescein injected intravenously (i.v,) was tested as a reference for NADH fluorometry of the cat cortex because (1) its fluorescence (peak: 520 nm; range: 500-550 nm) has been localized to retinal vessels during fluorescein retinal angiography 8, (2) it is excited to fluoresce by UV as well as by 450-500 nm light 3, (3) its fluorescence in small cortical blood vessels might be masked to some extent by increases in the blood volume of larger vessels and (4) its intensity would not be subject to reflected light artifacts. The fluorometric apparatus used in the present study was essentially the same as that described by Schuette et al. lo, where UV-illuminated tissue was viewed by a low light level television camera system, except for the following modifications: two wedge prisms were arranged in a holder fitted for two separate filter systems (Fig. 1) and this assembly was attached to the camera lens in place of the beam splitter assembly used in the earlier system. This enabled the presentation of two video images of equal optical

A simple video method for the quantification of microscopic objects

The design and operation of a simple, semi‐automatic video system for the analysis of geometric parameters of microscopic specimens is described. The system consists of standard video equipment and a custom built Electronic Integrator. The video signal from a camera attached to a microscope is mixed with the video signal from a second camera focused on a drawing board. A contour outline of the specimen to be analysed is drawn with the aid of a video monitor display of the combined images. The contour image is passed into the Electronic Integrator and analysed automatically. Logic circuitry in the Electronic Integrator permits analysis of complex contours independent of their orientation in the video raster. The area or volume of complex, overlapping specimens with restricted grey scale range can be rapidly analysed. The Electronic Integrator also is suitable for densitometric and specimen motion analyses.

Heart motion video-tracking (radarkymography) in diagnosis of congenital and acquired heart disease

Abstract Heart motion video-tracking (radarkymography) is a technic for recording the movements of cardiovascular structures. Over 100 patients with a variety of congenital and rheumatic heart lesions were studied. Characteristic graphic linear tracings were obtained in patients with mitral valve disease, obstruction to the aortic outflow tract, ventricular aneurysms and coarctation of the aorta. Radarkymography offers certain advantages over electrokymography, which to date has been the major technic for recording motion of the cardiac silhouette.

Effects of brain stem stimulation on cortical NADH fluorescence, blood flow, and O2 consumption in the cat

A causal relationship has been suggested between the altered electrical cortical activity and the increases in both cortical metabolism and blood flow which follow experimental reticulocortical activation. To further study this hypothesis, cortical O2 consumption, NADH fluorescence, and blood flow were monitored simultaneously with aortic blood pressure and ECoG after the electrical stimulation of the pontomesencephalic reticular formation (MRF) in anesthetized cats. Reversible increases in blood pressure, cortical blood flow, and O2 consumption, and decreases in NADH fluorescence were induced by MRF stimulation. All these changes were linearly related to each other as a function of stimulus intensity; they were often but not always associated with an increase in the ECoG frequency. MRF stimulation after ganglionic suppression with hexamethonium chloride revealed a dissociation of the resulting changes in the ECoG from the changes in cortical blood flow and metabolism. In addition, pharmacologically induced acute elevations of blood pressure caused changes in the experimental parameters similar to those which followed MRF stimulation but without a significant effect on the ECoG. We conclude that (i) acute increases in cortical blood flow and oxidative metabolism after electrical MRF stimulation may be more related to the simultaneous elevations of blood pressure than to changes in the ECoG; and (ii) an acute increase in cortical blood flow may by itself stimulate cortical oxidative metabolism in the anesthetized cat.

The pupil dark response in normal volunteers

A new semi-automatic videopupillometer with a two-channel video integrator uses prismatic displacement of pupil images for binocular recording with a single infrared-sensitive video camera. The video integrator graphically records pupil area or diameter during testing and can reanalyze and plot information from videotape recordings of pupil responses. This new pupillometer was used to evaluate the pupil dark response in 178 eyes of 89 normal human volunteers (ages 18-75 years). The time course of pupil dilation was similar in volunteers of all ages, even though the average pupil area became progressively smaller with increasing age.

Human eosinophil stimulation promoter lymphokine: production by antigen stimulated lymphocytes and assay with a new electro-optical technique.

Abstract A human lymphokine, eosinophil stimulation promoter (ESP), was shown to be produced by mononuclear leukocytes in response to non-helminthic antigens. Supernatants of tuberculin-stimulated cultures of mononuclear leukocytes from tuberculinskin test reactive humans contained non-dialyzable ESP activity; ESP could not be demonstrated in the supernatants of tuberculin stimulated cultures from skin test negative donors. Thus, human ESP, like murine ESP, is a soluble lymphokine product of sensitized mononuclear cells. ESP activity was also demonstrated in supernatants of mononuclear leukocyte cultures stimulated with streptococcal antigens. ESP selectively enhanced migration of eosinophils. ESP production could be blocked by puromycin, and its activity was diminished by prior incubation with eosinophil-rich granulocytes. Correlation of antigen stimulated ESP activity with skin test reactivity establishes ESP as another in vitro correlate of delayed hypersensitivity in man. A video method of data collection and analysis is described which greatly facilitates in vitro studies of ESP activity. The method is applicable to the study of other lymphokines which effect cellular migration.

Measurement of the Rate and Velocity of Movement by Single Heart Cells in Culture

A new method for the quantification of velocity of movement of single muscle cells in culture is described. The method involves television monitoring of microscope images and video analysis to yield data in chart form. Recorder printout is linearly related to distance of cell movement. Two new modifications of techniques for measuring the number of muscle cell movements are also described. These include television video analysis and a light-sensitive field effect transistor (Photo-fet). Application of both techniques to the measurement of contractions of single beating heart cells is shown, and the chronotropic effect of norepinephrine is monitored with increasing concentrations of the drug. Application of video analysis to the inotropic effect of the same drug also is studied, and quantitative data for velocity of contraction and relaxation are determined in microns per second.