Regional Energy Reserves in Mouse Brain and Changes With Ischaemia and Anaesthesia*

Abstract

WHEN the blood supply to the brain is cut off, function may be maintained for a short time through utilization of the energy reserve. This reserve has only four major components, P-creatine, ATP, glycogen, and glucose. Consequently the rates of change of these four substances during brief periods of complete ischaemia should be a valid measure of metabolic rate. Studies of whole brain (LOWRY, PASSONNEAU, HASSELBERGER and SCHULZ, 1964) and nerve (STEWART, PASSONNEAU and LOWRY, 1965) indicate this to be true. Although this 'closed system' method of measuring metabolic rate is relatively cumbersome it avoids some of the uncertainties of other procedures based on in vitro measurements or blood flow. The closed system method should be particularly useful for application to small areas of the brain for which there are no other methods available. Even single neuronal elements should eventually be accessible to this tool. This is a report of the application of this method to four gross regions of mouse brain and three histologically defined layers of mouse cerebellum. Measurements were made both with normal mice and with mice depressed with phenobarbitone. To provide a background, experiments are also reported with whole brain from mice given different dosages of phenobarbitone. E X P E R I M E N T A LReagents. All enzymes were obtained from Boehringer and Sons through Calbiochem, Berkeley, Calif., except glycogen phosphorylase (Sigma Chemical Co., St. Louis) and ox heart lactate dehydrogenase (Worthington, Freehold, N.J.). The phosphorylase was used as purchased for glycogen determination but was recrystallized three of four times to free it of ATPase and Pi when used for measuring the latter. Glycogen was dialysed against 0.025 M-acetate buffer at pH 4.7 to free it of contaminating Pi.Preparation ofmaterial. Male white mice of a local strain weighing about 20 g were used. These were decapitated, and the heads frozen either immediately or after a measured time interval (20 or 30 sec). Rapid freezing was accomplished by violent agitation in Freon-12 that had been chilled to its freezing point (-150") with liquid N,. Subsequently the tissue was kept at -80" whenever possible until enzymes present had been removed or inactivated. For studies of whole brain, the frozen tissue was powdered and treated with HClO, in such a manner as to avoid thawing and consequent changes in substrate concentrations (LOWRY et al. , 1964). The entire brain rostra1 to the inferior colliculi was used in this case. For the gross regional studies, 5 mg samples were dissected at -15" from four areas of the frozen brain with great care

Brain metabolism in uremic and adenosine-infused rats

“…Table III compares the present data with comparable published data. It is likely that the difference between the present results and those of Lowry et al (9)(10)(11) are largely due to the smaller head size in the mouse and differences in freezing technique.…”

“…The high brain glucose in the uremic rat merely reflects the fact that a slower rate of glucose consumption will permit brain glucose to more closely approach blood glucose during life. A marked increase of brain glucose is also seen in anesthesia and the extent of this increase has led others to suggest that, in addition to a decreased glucose consumption in anesthesia, there may be a primary increase of transport of glucose into brain (10,11). An increase in the extracellular fluid volume of brain in anesthesia and uremia could also produce a high brain glucose.…”

Subcellular Biochemical Investigation of Purkinje Neurons Using Synchrotron Radiation Fourier Transform Infrared Spectroscopic Imaging with a Focal Plane Array Detector

“…In particular, animal anesthesia, and the time period following animal death greatly affect localized lactate concentration. 21,70 Therefore, the lactate observed within the Purkinje neuron in this investigation may not reflect an in vivo state, but rather one induced during animal anesthesia and death. A thorough investigation of how sample preparation effects lactate concentration, and other biochemical markers effected by hypoxia-ischemia, which can be detected with novel spectroscopic techniques (i.e., FTIR, XAS, XRF), has been undertaken by this research group, and will be reported in a separate manuscript.…”

Anoxia in Mice: Reduced Glucose in Brain with Normal or Elevated Glucose in Plasma and Increased Survival after Glucose Treatment

“…ATP, P-creatine, and glucose were assayed by the methods of Lowry et al [lo]. Lactate and glycogen were measured by the methods of Gatfield et al [4]. For the determination of concentrations of glucose in the blood, samples of heparinized plasma were diluted 10-20 times in 0.5 M perchloric acid and centrifuged at 4".…”