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Edward Morley was an early member of the faculty of Western Reserve University in Cleveland. He was a very talented experimentalist who collaborated with Michelson of the Case School of Applied Science on the famous “Speed Of Light through the Ether” measurements in the 1880’s. In the 1890’s, Morley accomplished the difficult feat of providing the atomic weight of Oxygen, demonstrating by three separate quantitative methods and to the third decimal, that the atomic weight of oxygenwas 15.879, significantly less than the 16 predicted by the then current “Prout Hypothesis.”

A co-morbidity of sleep-disordered breathing is hypertension associated with elevated sympathetic nerve activity, which may result fromchronic intermittent hypoxia (CIH). CIH evokes plasticity in cardiorespiratory regulating sites, including the paraventricular nucleus (PVN), which acts to sustain increased sympathetic nerve activity. Our working hypothesis is that vasopressin neurons mediate the sustained increase in blood pressure and altered breathing associated with CIH. In a series of neuroanatomical experiments, we determined if vasopressin-containing PVN neurons innervate rostral ventrolateral medulla (RVLM), and altered cardiorespiratory responses induced by CIH conditioning (8h/day for 10 days) is mediated by vasopressin-V1A receptor signaling in the medulla. In the first set of experiments, cholera toxin β subunit was microinjected into the RVLM to delineate innervation of the PVN. Immunohistochemistry data showed vasopressin-containing PVN neurons were double-labeled with cholera toxin β subunit, indicating vasopressin projection to the RVLM. In the second set, sections of the medulla were immunolabeled for vasopressin V1A receptor, and its expression was significantly higher in the RVLM and in the neighboring rostral ventral respiratory column in CIH- than from RAconditioned rats. In a series of physiological experiments,we determined if blocking the vasopressin V1A receptor in the medulla would normalize blood pressure in CIHconditioned rats and also attenuate the evoked responses to PVN disinhibition.Blood pressure, heart rate, diaphragmatic and genioglossus muscle activity were recorded in anesthetized, ventilated and vagotomized rats. The PVN was disinhibited by microinjecting bicuculline before and after blocking vasopressin V1A receptors in the RVLM/rostral ventral respiratory column. In RA-conditioned rats, PVN disinhibition increased blood pressure, heart rate, minute diaphragmatic and genioglossus muscle activity, and these increases were attenuated after blocking the vasopressin V1A receptor. In CIH-conditioned rats, a significantly greater dose of blocker was required to blunt these physiological responses and it also normalized the baseline blood pressure. Our findings indicate that vasopressin is the neuropeptide released from PVN neurons that modulates cardiorespiratory output via the RVLMand rostral ventral respiratory column.

We1 have developed a reliable experimental method for measuring local regional cerebral blood flows in anesthetized mice. This method is an extension of the well-established single-pass dual-label indicator method for simultaneously measuring blood flow and glucose influx in rat brains. C57BL6J mice (n = 10) were anesthetized and regional blood flows (ml/min/g) were measured using the radio-tracer method. To test the sensitivity of this method we used a mathematical algorithm to predict the blood flows and compared the two sets of results.Measured regional blood flows between 0.7 and 1.7 ml/min/g were similar to those we have previously reported in the rat. The predicted blood flows using an assumed linearly increasing arterial tracer concentration-versus-time profile (that is, a ramp) were similar to the valuesmeasured in the physiological experiments (R2 0.99; slope 0.91). Thus,measurements of local regional cerebral blood flow in anesthetized mice using a single-pass radio-tracer method appear to be reliable.

To define some of the specific cellular effects of chronic hypoxia on the small intestine, we measured the concentration of glucose transporter 2 (GLUT2) at two sites, the jejunum and ileum. Wister rats were subjected to 21-day normoxia (n=6) or to continuous 21-day hypobaric hypoxia approximately 0.5 ATM (n=5). Western blot analysis was performed and the abundance of GLUT2 protein was quantified as relative densitometric units and normalized to actin. GLUT2 content was similar in the jejunum and ileum under normoxic (jejunum = 0.65±0.13 mean±SD; ileum = 0.56±0.22 OD; mean difference 0.09, p=0.09) and hypoxic conditions (jejunum=0.56±0.14 OD mean±SD; ileum = 0.58±0.16; mean difference −0.01, p =0.42). GLUT2 decreased by 14% of the mean normoxic jejunal levels whereas ileal GLUT2 was slightly increased. A maximum decline in weight of 15% occurred at day 4 followed by a blunted rate of weight gain for rats in the hypoxic group. Thus, sustained exposure to hypobaric hypoxia reduced the availability of GLUT2 for glucose transport in the jejunum. Regulating small intestinal content of glucose transporters may be an important mechanism for tissue adaptation to chronic hypoxia. This finding provides initial insight into hypoxic tolerance of the gut to chronic hypobaric hypoxic exposure.

Exposure of rats to mild hypoxia initially increases cerebral blood flow (CBF) as much as two-fold which maintains the arterial oxygen delivery rate. Several days after continued hypoxia, CBF decreases toward its baseline level as the blood oxygen carrying capacity is increased through increased hemoglobin content [1]. Evidently, CBF regulation and the oxygen carrying capacity of blood are correlated. To quantitatively analyze the CBF control mechanisms associated with chronic hypoxia, a mathematical model was developed that describes the concentration dynamics of O2 and CO2 transport and metabolic processes in blood and brain tissue. In capillary blood, species transport processes were represented by a one-dimensional convection-dispersion model with diffusion between blood and tissue cells in the cortex and brain stem. Three possible control mechanisms for CBF in response to chronic hypoxia were analyzed: 1) local PO2 change in cerebral tissue; 2) reduced blood flow associated with elevated blood viscosity from increased Hct; 3) neurogenic input from O2 receptors in the brain stem. Our hypothesis is that increased PO2 in the brain stem is the signal for the return of CBF to its baseline condition. This PO2 increase results from an increase in arterial oxygen carrying capacity and a decrease in local energy metabolism. Model simulations quantify the relative contributions of each of these control mechanisms during 4 days of hypoxic exposure. These simulations are consistent with experimental data that show CBF returns to its baseline even though the cerebral cortical tissue remains hypoxic as indicated by increased levels of the transcription factor Hypoxia Inducible Factor-1 (HIF-1).

Aged rat brain is more sensitive to reperfusion injury induced by cardiac arrest and resuscitation. The mitochondrial respiratory chain, the major source of free radicals during reperfusion, is likely to be the target of lipid peroxidation. Previous work has shown a higher mortality and lower hippocampal neuronal survival in older rats. 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, was found to be elevated in cortex and brainstem after resuscitation. In this study we investigated the acute changes of mitochondrial function in aging rat brain following cardiac arrest and resuscitation; the effect of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) was also tested. Fischer 344 rats, 6 and 24-month old, were subjected to cardiac arrest (7-10 minutes) and allowed to recover 1 hour after resuscitation. Mitochondria of cortex and brainstem were isolated and assayed for respiratory function. Compared to their respective non-arrested control group, 1h untreated groups (both 6 month and 24 month) had similar state 3 (ADP-stimulated) but higher state 4 (resting state) respiratory rates. The respiratory control ratio (state 3/state 4) of cortex in the 1h untreated group was 26% lower than the non-arrested control group; similar results were found in brainstem. The decreased mitochondrial respiratory function was improved by PBN treatment. HNE–modified mitochondrial proteins were elevated 1h after resuscitation, with an evident change in the aged. Treatment with PBN reduced the elevated HNE production in mitochondria of cortex. The data suggest (i) there is increased sensitivity to lipid peroxidation with aging, (ii) mitochondrial respiratory function related to coupled oxidation decreases following cardiac arrest and resuscitation, and (iii) treatment with antioxidant, such as PBN, reduces the oxidative damage following cardiac arrest and resuscitation.

Regulation of brain metabolism and cerebral blood flow involves complex control systems with several interacting variables at both cellular and organ levels. Quantitative understanding of the spatially and temporally heterogeneous brain control mechanisms during internal and external stimuli requires the development and validation of a computational (mathematical) model of metabolic processes in brain. This paper describes a computational model of cellular metabolismin blood-perfused brain tissue,which considers the astrocyteneuron lactate-shuttle (ANLS) hypothesis. The model structure consists of neurons, astrocytes, extra-cellular space, and a surrounding capillary network. Each cell is further compartmentalized into cytosol and mitochondria. Inter-compartment interaction is accounted in the form of passive and carrier-mediated transport. Our model was validated against experimental data reported by Crumrine and LaManna, who studied the effect of ischemia and its recovery on various intra-cellular tissue substrates under standard diet conditions. The effect of ketone bodies on brain metabolism was also examined under ischemic conditions following cardiac resuscitation through our model simulations. The influence of ketone bodies on lactate dynamics on mammalian brain following ischemia is studied incorporating experimental data.

Transient global brain ischemia induced by cardiac arrest and resuscitation (CAR) results in reperfusion injury associated with oxidative stress. Oxidative stress is known to produce delayed selective neuronal cell loss and impairment of brainstem function, leading to post-resuscitation mortality. Levels of 4-hydroxy-2-nonenal (HNE) modified protein adducts, a marker of oxidative stress, was found to be elevated after CAR in rat brain. In this study we investigated the effects of an antioxidant, alpha-phenyl-tert-butyl-nitrone (PBN) on the recovery following CAR in the aged rat brain. Male Fischer 344 rats (6, 12 and 24-month old) underwent 7-minute cardiac arrest before resuscitation. Brainstem function was assessed by hypoxic ventilatory response (HVR) and HNE-adducts were measured by western blot analysis. Our data showed that in the 24-month old rats, overall survival rate, hippocampal CA1 neuronal counts and HVR were significantly reduced compared to the younger rats. With PBN treatment, the recovery was improved in the aged rat brain, which was consistent with reduced HNE adducts in brain following CAR. Our data suggest that aged rats are more vulnerable to oxidative stress insult and treatment with PBN improves the outcome following reperfusion injury. The mechanism of action is most likely through the scavenging of reactive oxygen species resulting in reduced lipid peroxidation.

Control of the glycolytic pathway is crucial for the adaptation of the cellular and organismal energy‐producing systems in development, plasticity, and environmental challenges. Lack of proper regulation of glycolysis may underlie the pathology of carcinogenesis and tumorigenesis as well as the decreased adaptability and plasticity in aging. Hypoxia‐inducible factor‐1 (HIF‐1) is a ubiquitous transcription factor that responds to hypoxia and a variety of other energy‐related molecules to coordinate the upregulation of the glycolytic enzymes and glucose transporters, thus specifying the glycolytic capacity of the cell.

We have used a relatively simple model of hypoxia that triggers adaptive structural changes in the cerebral microvasculature to study the process of physiological angiogenesis. This model can be used to obtain mechanistic data for the processes that probably underlie the dynamic structural changes that occur in learning and the control of oxygen availability to the neurovascular unit. These mechanisms are broadly involved in a wide variety of pathophysiological processes. This is the vascular component to CNS functional plasticity, supporting learning and adaptation. The angiogenic process may wane with age, contributing to the decreasing ability to survive metabolic stress and the diminution of neuronal plasticity.

We directly measured pHi using the pH sensitive dye, neutral red. We defined pHi for rectal and gastric tissue in whole tissue and by layer under control and arrest conditions. Fifteen minutes of arrest was not sufficient time to alter the pHi at the rectal or gastric site. On initial inspection, the stomach may be more sensitive to ischemic changes than the rectum. Understanding the mechanism by which PCO2 generation is used to track clinical changes is vital to the early detection of tissue dysoxia in order to effectively treat and manage critically ill patients.

The benefits of a mouse model are efficiency and availability of transgenics/knockouts. Quantitation of cerebral blood in small animals is difficult because the cannulation procedure may introduce errors. The [14C]-iodoantipyrine autoradiography (IAP) method requires both the tissue concentration and the time course of arterial concentration of the [14C] radioactive tracer. A single point-analysis technique was evaluated for measuring blood flow in mice (30 g ± 0.3 g; n=11) by using computational models of sensitivity analysis, which quantitates relationships between the predictions of a model and its parameters. Using [14C]-IAP in conjunction with mathematical algorithms and assumed arterial concentration-versus-time profiles, cortical blood flow was deduced from single-point measurements of the arterial tracer concentration. The data showed the arterial concentration profile that produced the most realistic blood flows (1.6 ± 0.4; mean ± SD, ml/g/min) was a profile with a ramp time of 30 sec followed by a constant value over the remaining time period of 30 sec. Sensitivity analysis showed that the total experimental time period was a more important parameter than the lag period and the ramp period. Thus, it appears that the accuracy of the assumption of linearly increasing arterial concentration depends on the experimental time period and the final arterial [14C]-iodoantipyrine concentration.

Recent evidence suggests that cytochrome oxidase is partially reduced under resting conditions in the brain. Previous data, recorded over the past 30 years from intact brain using optical methods in the visible wavelength range, are consistent with this observation. These older data, while not conclusive in themselves, support the overall conclusions. The historical perspective on the experiments and controversies illustrates a number of useful principles. The first is that new methods tend to produce new observations that may be difficult to reproduce due to the uniqueness of the instrumentation. The second is that any new and different observations cannot be assimilated without an acceptable theoretical framework and, without assimilation can have little impact. Finally, the mechanisms which might explain why cytochrome oxidase may be more reduced than previously thought are still not fully developed and, therefore, the physiological significance of such reduction is not known.

An acute reduction in oxygen delivery to a tissue is associated with metabolic changes aimed at maintaining ATP homeostasis. However, given the complexity of the human bioenergetic system, it is difficult to determine quantitatively how cellular metabolic processes interact to maintain ATP homeostasis during stress (e.g., hypoxia, ischemia, and exercise). In particular, we are interested in determining mechanisms relating cellular oxygen concentration to observed metabolic responses at the cellular, tissue, organ, and whole body levels and in quantifying how changes in tissue oxygen availability affect the pathways of ATP synthesis and the metabolites that control these pathways. In this study, we extend a previously developed mathematical model of human bioenergetics, to provide a physicochemical framework that permits quantitative understanding of oxygen as a metabolic regulator. Specifically, the enhancement--sensitivity analysis--permits studying the effects of variations in tissue oxygenation and parameters controlling cellular respiration on glycolysis, lactate production, and pyruvate oxidation. The analysis can distinguish between parameters that must be determined accurately and those that require less precision, based on their effects on model predictions. This capability may prove to be important in optimizing experimental design, thus reducing use of animals.

A common feature of senescent organisms is the decline in their ability to respond to different types of stress such as hypoxia or ischemia. Previously it was reported that aging was associated with a defect in compensatory angiogenesis in response to tissue ischemia. This deficit was associated to a reduction of vascular endothelial growth factor (VEGF) expression, an endothelial-specific mitogen that is essential for angiogenesis during development and adulthood. VEGF as well as other hypoxia-responsive genes share a common regulatory pathway. They are regulated by the transcription factor hypoxia inducible factor-1 (HIF-1), a heterodimeric protein consisting of two subunits, HIF-1a and HIF-113. Both subunits are required for DNA binding and transactivation of target genes. HIF-la is unique to HIF-1 and its expression is primarily regulated by oxygen tension. During normoxia the HIF-la gene is expressed continuously; however, the HIE-la protein is rapidly degraded by the ubiquitin-proteosome system. During hypoxia, degradation of HIF-la is suppressed and thereby it accumulates in the nucleus almost instantly. In contrast, the HIP-113 subunit is constitutively expressed and its levels do not change significantly during hypoxia

Hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression were studied in rat cerebral cortex after reversible global cerebral ischemia produced by cardiac arrest and resuscitation. Immunoblot analysis showed a significant induction of HIF-1α protein after 1 hour of recovery from cardiac arrest which remained elevated for at least 12 hours. Upregulation of VEGF mRNA and protein were also observed but this was delayed in comparison to the HIF- 1α response. VEGF188 and VEGF164 mRNA levels were increased at 12-48 h of recovery from cardiac arrest but returned to basal expression after 7 days. Changes in VEGF120 mRNA expression did not reach statistical significance. Correspondingly, VEGF protein levels increased by about double at 24 and 48 hours of recovery but returned to basal levels after 7 days. These results suggest that cardiac arrest and resuscitation triggers HIF-1α induction, which might be at least in part responsible for the stimulation of VEGF expression.

Angiogenesis is defined as the formation of new vascular networks from pre-existing blood vessels. The mature vascular network of the adult is relatively stable and vascular endothelial cells exist in a differentiated, quiescent state and rarely proliferate. Angiogenic remodeling occurs only in certain situations, hypoxia and ischemia being primary inducers of angiogenesis. In the brain, angiogenesis is a critical compensatory strategy for brain adaptation to chronic hypoxia, which presumably results in shorter intercapillary diffusional distances and improvement of tissue oxygenation. This adaptive response is reversible upon return to normoxia, although the mechanism controlling vascular regression is not known.

Uncoupling protein 5 (UCP5), also referred to as brain mitochondrial carrier protein (BMCP 1), belongs to the family of mitochondrial membrane transporters known as uncoupling proteins (UCPs)1. Five UCPs have been cloned, named UCP1, UCP2, UCP3, UCP4 and UCP5/BMCP12. It is well established that UCP1, the prototypical UCP expressed only in brown adipocytes, dissipates the mitsochondrial proton gradient across the inner membrane and hence potential energy is lost as heat2. However, it is not known whether UCP 2–5 are true uncoupling proteins and have thermogenic properties or have other in vivo physiological functions. It has been proposed that the novel UCPs might play a role in the regulation of reactive oxygen species (ROS) production3,4

Cardiac arrest and resuscitation produce global cerebral ischemia and reperfusion damage to the brain, which lead to high mortality and delayed neuronal death. Adenosine (ADO) has been suggested as an endogenous neuroprotective molecule, acting through multiple potential mechanisms (Sweeney, 1997). In this study we investigated the possible neuroprotective effects of adenosine on cerebral recovery following global ischemia induced by cardiac arrest and resuscitation.

The central inhibitory effect of systemic hypoxia on respiration can be explained by different mechanisms including withdrawal of excitatory inputs to respiratory related neurons. It has been shown that several groups of catecholamine containing neurons, including the locus ceruleus (LC), express chemosensitive properties. Different stimuli like hypercapnia, hypoxia or electrical stimulation of carotid sinus nerve, induce c-fos protein expression in the LC neurons.1,2 Furthermore, their activation by increased hydrogen ions causes elevation of respiratory drive.3

Nitric oxide (NO) is a remarkable biological messenger that participates in a wide range of functions in the central and peripheral nervous systems. Since this free-radical gas cannot be stored in synaptic vesicles like other neurotransmitters, the activity of nitric oxide synthase must be carefully modulated to provide appropriate levels of NO.

The energy requirements of the brain are large and immediate. The need to deliver adequate oxygen and glucose and to remove carbon dioxide specifies the vascular architecture. Taken as a whole, the metabolic demand of the brain is fairly constant during natural functions including wakefulness and sleep. Matched against this constant central demand is the varying cardiac output that serves the changing systemic needs that vary with physical activity. This major autoregulatory function occurs at the level of the larger brain vessels which act to keep brain blood flow constant despite wide systemic swings in cardiac output and blood pressure. Although overall brain blood flow is relatively constant, local and regional brain flow must be characterized as both spatially and temporally heterogeneous. An additional important constraint is that central nervous system neurons are vulnerable to oxidative damage from reactive oxygen species, and, therefore, lower exposure to oxygen is more compatible with long term continuance of function. Thus, normal brain function appears to occur with very low levels of tissue oxygen in brain regions that are not active. Once activated, however, blood flow to these regions becomes elevated while activity persists. The challenge for the cerebrovascular control mechanisms, then, is to maintain a low oxygen cellular milieu during quiescence, but rapidly provide a high oxygen supply during transient periods of neuronal activation. This latter task becomes more difficult in a low ambient oxygen environment.

It is well known that hypoxia induces increased respiration through activation of peripheral chemoreceptor reflex pathways. But, in the absence of afferent inputs from peripheral chemoreceptors, systemic hypoxia causes depression of breathing activity. Evidence of hypoxic depression of respiration also can be observed in animals and in humans with intact peripheral chemoreceptors.The effects of central hypoxia on respiratory activity require more time to develop than the effects of hypoxic stimulation through peripheral chemoreceptors, suggesting the involvement of relatively slow metabolic processes. The mechanism by which hypoxia causes central depression of breathing is not known, but several possibilites can be suggested.

Chronic exposure to moderate hypoxia is associated with considerable remodeling of the cerebral microvascular network. In rats exposed to hypobaric (0.5 ATM) hypoxia, cortical microvessel density increases to 171% of controls within the first week. Peak density is reached within two weeks. The increase in hematocrit follows a similar time course. Although the hemodynamic acclimation appears to complete after two weeks, levels of cortical metabolites, obtained after three weeks of exposure, suggest O2 insufficiency at rest. Increased lactate and slightly decreased glycogen concentrations within the brain, along with increased glucose consumption are consistent with an increased dependence on anaerobic glycolysis for energy production. Whatever the production mechanisms, the restored ATP and phosphocreatine levels indicate the energy demand is being met.

Near-infrared spectrophotometry (NIR) is a non-invasive measure of tissue oxygen delivery and utilization1. Its use has been limited, with one notable exception2, to subjects at rest under conditions of ischemia. It is not clear whether reproducible and accurate measures of hypoxia using this technique can be obtained during dynamic exercise in the upper extremity, because changes in optical path length can alter the signals. The technique’s greatest potential power as a clinical diagnostic tool in abnormalities of oxygen transport and utilization lies in the responses to changes in oxygen consumption, i.e. exercise.

Hydrogen ions play an important role in cellular processes. There are intimate links between energy metabolism and the control of cell and tissue acid/base balance. Control of this balance is threatened or lost during severe hypoxia or ischemia. Re-establishment of pH balance must occur before the tissue can be considered to have returned to normal operating conditions. Because hydrogen ions influence so many reactions, the timing of renormalization can be crucial to the entire recovery process. Indeed, in many active tissues, too fast reversal of acidosis during recovery from severe hypoxia or ischemia appears to be detrimental to the overall recovery of homeostasis. That a tissue could restore function more rapidly if mild acidosis were maintained during the immediate post-stress recovery time has been referred to as the “pH paradox” (Currin et al., 1991), in analogy with the so-called “calcium paradox” that has been discussed primarily in the cardiovascular literature. In this paper we will review the changes that occur in pHi during hypoxia and ischemia in rat brain. We will explore the interrelationships of protons with metabolism, and we will propose a scheme for the interaction of protons in brain function. Finally, we will attempt to reach a conclusion concerning the applicability of the concept of pH paradox in brain.

Continued exposure of rats to hypobaric hypoxia results in both functional and structural changes in brain. A most striking structural change is the 50-80% increased capillary density in the cerebral cortex and other brain regions after 3 weeks of hypoxic exposure (LaManna et al., 1992; LaManna, 1992). The apparent increased density may be accompanied by a shift in the capillary segment length distribution towards longer capillary segments (LaManna et al., 1993). It is still not clear if the increased capillary density is due to new capillary branching or to elongation of existing capillaries, or both. Nevertheless, it seems likely that the microvascular adaptation to continued hypoxia is controlled by growth factors. The growth factors that control angiogenesis are not yet well understood, but basic fibroblast growth factor (bFGF) is known to activate endothelial cells resulting in capillary growth (de Juan et al., 1990), and this growth factor is increased following metabolic stress such as brain ischemia (Finkelstein et al., 1990; Lyons et al., 1991). In this study, we determined whether bFGF mRNA was increased in brains of rats exposed to 3 weeks of hypoxia.

The common mammalian physiological response to moderate hypoxia, such as experienced with exposure to altitude up to about 5000m, initially includes increased ventilation and increased cerebral blood flow, followed by elevated hemoglobin concentration (Dempsey and Forster, 1982). We have previously shown that rats follow this usual pattern (Shockley and LaManna, 1988; LaManna et al., 1984; LaManna et al., 1989; LaManna et al., 1992; LaManna, in press). Rats adapt to moderate hypoxia by increased ventilation, increased blood hemoglobin and failure to gain weight (LaManna et al., 1992). For the brain, the most dramatic response to continued hypoxia was an increase in capillary density, resulting in decreased intercapillary distance achieved through angiogenesis that allows brain tissue oxygen diffusion flux to remain adequate. This structural plasticity is accompanied by functional changes as indicated by the increased glucose transporter density observed in the hypoxic adapted rat brain (Harik et al., 1991). The purpose of this present study was to begin to characterize any changes in the geometry of the microvascular capillary network resulting from hypoxic induced angiogenesis.

Recent developments in dye chemistry and modern optics have made simultaneous measurement of multiple intracellular ion species possible1. Analysis of optical spectra from intact tissues is complicated because of the presence of multiple components. Quan­titative descriptions of these components are required to apply these techniques to analyti­cal chemistry and cellular physiology. Ratio methods2 have been applied to many areas of quantitative optical signal measurement because the quantitative data is independent of dye concentration, path length and light source intensity. However, it is known that the ratio method can give erroneous results3 without special attention to the choice of the op­timal wavelengths for these methods. Full spectra carry all the information needed for qualitative and quantitative analysis. Methods like principal component regression (PCR) and partial least-squares (PLS), which have been used for full spectra calibration in most chemometrics literature4,5, or multicomponent stripping for image applications 6,7,8 need heavy computation times to perform their optimization processes.

Brain lactate is elevated in certain pathophysiological conditions such as hypoxic or ischemic brain injury, and plasma lactate levels are elevated during conditions of systemic metabolic stress. In either case, the transport of lactate at the blood-brain barrier (BBB) may play an important role in determining eventual tissue survival. BBB lactate transport is thought to occur via a facilitative, non-concentrative diffusion mechanism (Nemoto et al., 1974; Oldendorf, 1972; Oldendorf, 1973; Gjedde et al., 1975) which is linked to the monocarboxylic acid transporter that also transports pyruvate (Oldendorf, 1973; Pardridge and Oldendorf, 1977). Nevertheless, it is unclear from the previous reports whether the capacity of this system to transport lactate would be functionally meaningful. Here, we measured the rate of lactate influx into brain at plasma concentrations encountered during pathological conditions.

Stroke in humans is a syndrome consisting of the abrupt onset of a focal neurological deficit. The etiology of stroke is varied, but the majority of strokes are due to brain infarction from arterial occlusion (Walker et al., 1981; Robins and Baum, 1981). The rest are attributed to infarction, intracerebral hemorrhage, and subarachnoid hemorrhage. Experimental focal ischemia produced by intracranial vessel occlusion closely resembles the clinical manifestations of cerebral ischemia presenting as stroke. The presence of different perfusion zones following vessel occlusion is responsible for the unique characteristics of focal, as opposed to global, ischemia. The variability of the collateral circulation and of tissue perfusion renders the experimental investigation of focal ischemia inherently more difficult than the investigation of global ischemia. The complexity of focal stroke is further compounded by an incomplete understanding of the control mechanisms regulating blood flow of the brain under normal and pathological conditions. Our working hypothesis is that the normal coupling mechanisms among function, blood flow, and metabolism are perturbed during and after ischemia and, furthermore, that the pathophysiologies in the focal and perifocal regions are distinct. For this reason, the results discussed in this chapter are prefaced by an overview of the physiology of energy metabolism and blood flow in the brain. The chapter will then briefly review the clinical significance of stroke in an attempt to underscore the importance of, and the problems with, focal ischemia models; finally, the biochemical correlates to brain injury following irreversible focal ischemia will be described in some detail.

The normal function of the brain is entirely dependent on an adequate supply of oxygen for the provision of ATP via oxidative energy metabolism. But, the presence of oxygen in excess of metabolic needs can be toxic, with especially drastic consequences for an organ such as the brain in which neurons cannot be regenerated once lost. Thus, there are mechanisms that have evolved, apparently in response to these opposing requirements, which control oxygen delivery to the brain so that it is tightly coupled to tissue oxygen consumption, such that energy demand is satisfied but brain oxygen exposure is minimized. This principle of just sufficient oxygen implies that under normal ambient oxygen pressures and normal physiological conditions, regional brain blood flow is relatively low and brain tissue oxygen tension is also low (Sick et al., 1982). Increases in functional energy demand would have to be accompanied by at least transient increases in local oxygen delivery (LaManna et al., 1987).

The carbonic anhydrase inhibitor, acetazolamide (Maren, 1967), has been used clinically for many years as a treatment for the prevention of the symptoms, primarily headaches, of acute exposure to altitude hypoxia, as originally demonstrated by Cain and Dunn (1966). This effect was thought to be due to a drug-induced metabolic acidosis that compensated for an altitude-induced respiratory alkalosis. Furthermore, it had already been noticed that acetazolamide administration was followed by increased cerebral blood flow (Cotev et al., 1968; Ehrenreich et al., 1961; Posner and Plum, 1960), an observation that has often been confirmed since. Nevertheless, the effects of acetazolamide on metabolism and blood flow in the brain and their relationships to the physiological mechanisms of action of acetazolamide have not been well studied.

Regulation of intracellular pH (pHi) is a critical factor in brain function in normal and pathological states. An inability to maintain controlled pHi during and after conditions which impose severe metabolic stress on the brain has been implicated as contributory to permanent neurologic damage in severe hypoxia, ischemic stroke, and seizures (1,2). The relationship between energy metabolism and pHi has been difficult to study because of spatial and temporal heterogeneity in brain physiology. Furthermore, there is a lack of effective techniques for determining pHi in brain which are compatible with concurrent measurement, or control, of other variables which affect or reflect cell physiology. For example, tissue lactate levels have been considered to reflect pHi, but this relationship has not been verified by direct experimental determination. The quantitative method for determining tissue lactate, which also preserves spatial distribution, excludes most existing techniques for quantitative pH measurement. This problem might be resolved by the use of colorimetric pH indicator dyes. The requirements for an in vivo colorimetric pH indicator in the mammalian central nervous system are stringent, but can be met by the vital dye neutral red (3).

A full understanding of the delivery of oxygen to a tissue cannot be attained by study of the blood flow to that tissue alone. An important component of the delivery system is the particular spatial arrangement of the capillaries supplying the tissue. This is especially critical in capillary networks that function through dynamic control of local tissue perfusion by regulating the number of open and closed capillaries. This form of vascular regulation has been generally known as ‘capillary recruitment’ and this concept implies that there are dynamic changes in tissue blood volume in response to changes in tissue metabolic demand. Capillary recruitment has long been suspected of being an integral part of cerebrovascular function, but until recently, experimental evidence for significant participation has been lacking (Weis et al., 1982; Pardridge and Fierer, 1985).

The brain is exquisitely sensitive to changes in its oxygen supply. Indeed, oxygen delivery to the brain is regulated on a moment to moment basis. While the brain depends on oxygen for oxidative energy metabolism, there is also the danger of excess exposure to oxygen and the attendant problems of oxygen toxicity. Consequently, there must be mechanisms which regulate regional cerebral blood flow that optimize the contradictory requirements of energy demand and oxygen toxicity. Thus, regional blood flow is usually kept low so that brain tissue oxygen tension is low (Sick et al., 1982) to avoid toxicity until functional requirements produce increased neuronal activity which are accompanied by local increases in blood flow to accommodate the resultant increased energy demand (Kreisman et al., 1981; laManna et al., 1987). This hypothesis is a possible explanation for the paradoxical observations which indicate that the brain is always “on the brink” of hypoxia (Rosenthal et al., 1976) despite a demonstrable large reserve blood flew capacity. The mechanisms of local control of oxygen delivery seem to be set so that there is just sufficient oxygen for immediate energy demand but no more. Hypoxic and hypoxemic conditions will obviously force a response from the regulatory mechanisms in order that the balance between oxygen delivery and consumption be maintained (LaManna et al., 1984).

About 25 years ago, major breakthroughs were made in our understanding of the neurochemistry of the basal ganglia and the biochemical pathology of Parkinson’s disease, which eventually led to the successful introduction of levodopa as replacement therapy for this malady. This dramatic success story, especially after the subsequent introduction of peripheral DOPA decarboxylase inhibitors and dopamine receptor agonists as adjuvants, was hailed as one of the best examples of how basic biomedical research can lead to rational therapies for human diseases. Meanwhile, the cause of Parkinson’s disease, i.e. the progressive degeneration of nigrostriatal dopaminergic neurons, remained unknown. Furthermore, replacement therapy, which was once considered revolutionary, was found to be of little help in the treatment of subjects with advanced Parkinsonism and entirely ineffective in arresting the inexorable progression of Parkinson’s disease. Another important fact that emerged in the last decade concerns the lack of evidence that Parkinson’s disease is a genetic disorder, as evidenced by the absence of increased concordance among identical twins. This led to the belief that it may be caused by environmental factors (if it is not nature, it must be nurture).

The delivery of oxygen and glucose to the cerebral cortex is tightly matched to energy demand under a wide variety of physiologic and probably under some pathophysiologic conditions. The maintenance of this relationship must involve the ability to detect changes in metabolic requirements and to modify the delivery of oxygen and substrates through a delicate control of cerebral blood flow (CBF). Furthermore, since many physiologic and pathophysiologic conditions cause only regional alterations in cerebral metabolic activity, tight matching of supply and demand will have to act at a local, regional or global level.

Because of its high oxidative metabolic rate, the brain is a significant heat producer. As with all mammalian tissue, the operating temperature range of the brain must be kept at a nearly constant level for normal function. Brain function is especially sensitive to increases in temperature (Cabanac, 1986). The temperature of the brain tissue is determined by the heat conduction properties of the tissue, the heat produced by metabolism, and the heat and heat-transfer capacity of the blood flowing through the tissue. It has always been thought that one of the functions of the cerebral circulation was to act as a cooler of the brain, and some mammalian species have a carotid rete system to maximize this cooling effect (Baker, 1979; Cabanac, 1986). While there is data on the larger animals such as dog, cat, and primate which indicate that significant cooling of the brain during physical excercise occurs (Baker, 1979; Hayward, 1966), there is only sparse information concerning the relationships of metabolism and blood flew in the CNS of smaller mammals such as rodents.