Karl A. Kasischke

Cortical spreading depression causes and coincides with tissue hypoxia

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that has been implicated in migraine and in progressive neuronal injury after stroke and head trauma. Using two-photon microscopic NADH imaging and oxygen sensor microelectrodes in live mouse cortex, we find that CSD is linked to severe hypoxia and marked neuronal swelling that can last up to several minutes. Changes in dendritic structures and loss of spines during CSD are comparable to those during anoxic depolarization. Increasing O2 availability shortens the duration of CSD and improves local redox state. Our results indicate that tissue hypoxia associated with CSD is caused by a transient increase in O2 demand exceeding vascular O2 supply.

Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions

Oxygen transport imposes a possible constraint on the brains ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO2 range with a p 50 of 3.4±0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution.

Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease

Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle‐specific expression of a novel mitochondrial‐targeted superoxide biosensor (mt‐cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin‐D‐mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3±2.0 flashes/1000 μm2·100 s before and after 5 tetani) and markedly decreased (to 7.7±1.6 flashes/1000 μm2 ·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature‐dependent increase in mSOF frequency (11.9±0.8 and 19.8±2.6 flashes/1000 μm2 ·100 s at 23°C and 37°C) was observed in fibers from RYR1Y522S/WT mice, a mouse model of malignant hyperthermia and heat‐induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress.—Wei, L., Salahura, G., Boncompagni, S., Kasischke, K. A., Protasi, F., Sheu, S.‐S., Dirksen, R. T. Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease. FASEB J. 25, 3068–3078 (2011). www.fasebj.org

In Vivo Imaging of Cerebral Energy Metabolism with Two-Photon Fluorescence Lifetime Microscopy of NADH

Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

HIV-1 Trans Activator of Transcription Protein Elicits Mitochondrial Hyperpolarization and Respiratory Deficit, with Dysregulation of Complex IV and Nicotinamide Adenine Dinucleotide Homeostasis in Cortical Neurons

HIV-1 causes a common, progressive neurological disorder known as HIV-associated dementia (HAD). The prevalence of this disorder has increased despite the use of highly active antiretroviral therapy, and its underlying pathogenesis remains poorly understood. However, evidence suggests that some aspects of HAD may be reversible. To model the reversible aspects of HAD, we have used the HIV-1 neurotoxin trans activator of transcription protein (Tat) to investigate nonlethal changes in cultured neurons. Exposure of rodent cortical neurons to sublethal concentrations of Tat elicits mitochondrial hyperpolarization. In this study, we used the cationic lipophilic dye rhodamine 123 to confirm this observation, and then performed follow-up studies to examine the mechanism involved. In intact neurons, we found Tat elicited a rapid drop in internal mitochondrial pH, and addition of Tat to purified mitochondrial extracts inhibited complex IV of the electron transport chain. To correlate enzyme activity in mitochondrial extracts with results in intact cells, we measured neuronal respiration following Tat exposure. Cortical neurons demonstrated decreased respiration upon Tat treatment, consistent with inhibition of complex IV. We examined mitochondrial Ca2+ homeostasis using a mitochondrial targeted enhanced yellow fluorescent protein-calmodulin construct. We detected a decrease in mitochondrial calcium concentration following exposure to Tat. Finally, we measured the energy intermediate NAD(P)H after Tat treatment, and found a 20% decrease in the autofluorescence. Based on these findings, we suggest that decreased NAD(P)H and calcium concentration contribute to subsequent respiratory decline after exposure to Tat, with detrimental effects on neuronal signaling.

Excess soluble CD40L contributes to blood brain barrier permeability in vivo: implications for HIV-associated neurocognitive disorders.

Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L) are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB) permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat) can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1) positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking.

Methamphetamine causes sustained depression in cerebral blood flow

The use prevalence of the highly addictive psychostimulant methamphetamine (MA) has been steadily increasing over the past decade. MA abuse has been associated with both transient and permanent alterations in cerebral blood flow (CBF), hemorrhage, cerebrovascular accidents and death. To understand MA-induced changes in CBF, we exposed C56BL/6 mice to an acute bolus of MA (5mg/kg MA, delivered IP). This elicited a biphasic CBF response, characterized by an initial transient increase (~ 5 minutes) followed by a prolonged decrease (~ 30 minutes) of approximately 25% relative to baseline CBF--as measured by laser Doppler flowmetry over the somatosensory cortex. To assess if this was due to catecholamine derived vasoconstriction, phentolamine, an α-adrenergic antagonist was administered prior to MA treatment. This reduced the initial increase in CBF but failed to prevent the subsequent, sustained decrease in CBF. Consistent with prior reports, MA caused a transient increase in mean arterial blood pressure, body temperature and respiratory rate. Elevated respiratory rate resulted in hypocapnia. When respiratory rate was controlled by artificially ventilating mice, blood PaCO(2) levels after MA exposure remained unchanged from physiologic levels, and the MA-induced decrease in CBF was abolished. In vivo two-photon imaging of cerebral blood vessels revealed sustained MA-induced vasoconstriction of pial arterioles, consistent with laser Doppler flowmetry data. These findings show that even a single, acute exposure to MA can result in profound changes in CBF, with potentially deleterious consequences for brain function.

Primary hypoxic tolerance and chemical preconditioning during estrus cycle in mice.

BACKGROUND AND PURPOSE Exogenous application of estrogens or progesterone ameliorates hypoxic/ischemic cell damage. This study investigates whether values of primary and induced hypoxic tolerance vary endogenously during the estrus cycle in female mice. METHODS Population spike amplitude (PSA) and NADH were measured during hypoxic hypoxia and recovery in hippocampal slices from untreated control animals (C slices) and slices prepared from animals pretreated in vivo with a single intraperitoneal injection of 3-nitropropionate (3NP) (3NP slices) or acetylsalicylate (ASA) (ASA slices). RESULTS Posthypoxic recovery of PSA was dose dependent in 3NP slices from males, with maximal recovery on pretreatment attained with 20 mg/kg 3NP (82+/-32% [mean+/-SD]; C slices, 38+/-29%; P<0.01). PSA recovered to 17+/-12% in C slices during proestrus, 43+/-23% during estrus, and 63+/-44% during diestrus. In 3NP slices, recovery of PSA increased to 57+/-36% (P<0. 05) during proestrus. Hypoxic tolerance was not increased in other stages of the estrus cycle. Hypoxic NADH increase during proestrus declined from 212+/-76% in C slices to 133+/-11% in 3NP slices (P<0. 05). Recovery of PSA in ASA slices was 75+/-36% (P<0.01 versus control) in males and 48+/-34% during proestrus (P<0.05 versus ASA slices from males). CONCLUSIONS Primary and induced hypoxic tolerance are endogenously modulated during the estrus cycle. Differences in hypoxic oxidative energy metabolism mediate part of the differential tolerance. Experimental and clinical therapeutic strategies against cerebral ischemia/hypoxia need to consider sex-related dependence.

Two-photon microscopy of cortical NADH fluorescence intensity changes: correcting contamination from the hemodynamic response

Quantification of nicotinamide adenine dinucleotide (NADH) changes during functional brain activation and pathological conditions provides critical insight into brain metabolism. Of the different imaging modalities, two-photon laser scanning microscopy (TPLSM) is becoming an important tool for cellular-resolution measurements of NADH changes associated with cellular metabolic changes. However, NADH fluorescence emission is strongly absorbed by hemoglobin. As a result, in vivo measurements are significantly affected by the hemodynamics associated with physiological and pathophysiological manipulations. We model NADH fluorescence excitation and emission in TPLSM imaging based on precise maps of cerebral microvasculature. The effects of hemoglobin optical absorption and optical scattering from red blood cells, changes in blood volume and hemoglobin oxygen saturation, vessel size, and location with respect to imaging location are explored. A simple technique for correcting the measured NADH fluorescence intensity changes is provided, with the utilization of a parallel measurement of a physiologically inert fluorophore. The model is applied to TPLSM measurements of NADH fluorescence intensity changes in rat somatosensory cortex during mild hypoxia and hyperoxia. The general approach of the correction algorithm can be extended to other TPLSM measurements, where changes in the optical properties of the tissue confound physiological measurements, such as the detection of calcium dynamics.

A new pathway for lactate production in the CNS

Lactate has long been considered to be a potentially damaging final metabolite of anaerobic glycolysis and has received little interest by neuroscientists. Interest in lactate began to increase with the demonstration of non-oxidative glucose consumption (i.e. glycolysis) in the activated brain by a landmark PET study (Fox et al. 1988). Numerous studies have since measured lactate with the consensus that sustained focal neural activity in the CNS is inevitably accompanied by an increase in extracellular lactate (Prichard et al. 1988; Mangia et al. 2007). In this context, Pellerin & Magistretti (1994) proposed that lactate may not be a metabolic dead-end product but rather the dominant oxidative substrate for neurons. The formulation of the astrocyte–neuron lactate shuttle hypothesis has generated tremendous interest in the cellular source and fate of lactate, to the point that ‘lactate’ has become a contentious term and is frequently associated with academic disputes. In this issue of The Journal of Physiology, Caesar et al. (2008) open a new perspective on the generation of extracellular lactate. As is often the case, this intriguing study advances the field but also raises more questions than it answers. Since the original report by Pellerin & Magistretti, it has been widely assumed that lactate production takes place in astrocytes and is triggered by astrocytic glutamate transporters. In stark contrast to this view, Caesar et al. provide evidence that lactate production is the consequence of AMPA receptor activation. They report in vivo microdialysis measurements of extracellular lactate concentration in the molecular layer of rat cerebellum. These measurements are interpreted in the context of changes in cerebral blood flow, electrical activity, tissue oxygen tension and regional glucose utilization. The study design is straightforward: sustained climbing fibre stimulation at 5 Hz induces neural activity in Purkinje cells with a robust rise in extracellular lactate and a correlated rise in oxygen consumption in the molecular layer of the cerebellum. The effect of AMPA receptor blockade is investigated. According to the prevailing view that lactate production is triggered by astrocytic glutamate uptake, CNQX should have no effect. Surprisingly, climbing stimulation under CNQX did not lead to an observable rise in lactate. The wide-reaching conclusion is that lactate production in the cerebellar cortex is mediated by AMPA receptors rather than glutamate transporters. Importantly, Duan et al. (1999) have previously shown that CNQX has no effect on astrocytic glutamate uptake. Consequently, the proposed glutamate transporter-mediated pathway is left intact. With the proposal of a glutamate transporter-independent pathway and the possibility of a postsynaptic site for lactate production, the paper by Caesar et al. provides a double challenge to the lactate shuttle hypothesis. Nevertheless, the authors overtly state that their study still respects the possibility of lactate production and shuttling from Bergmann glia to neurons. Indeed, AMPA receptors are present on both neuronal (Purkinje cells) and glial cells (Bergmann astrocytes) in the cerebellar cortex. Consequently, the current study which relies on CNQX blockade of AMPA receptors alone cannot answer the pressing question of whether lactate production is of neuronal or glial origin. Future studies using NASPM, an antagonist of Ca2+-permeable AMPA receptors expressed by Bergmann glia may clarify this issue. While CNQX clearly abolishes transient lactate increases, it should be considered that CNQX essentially silences all measured responses in the cerebellar cortex with loss of electrical activity, the blood-flow response, tissue oxygen use and glucose uptake (Offenhauser et al. 2005; Caesar et al. 2008). This of course does not prove that all these processes are mediated by AMPA receptors. To effectively rule out (or verify) glutamate transporters as mediators of lactate secretion, direct manipulations of glutamate transporter activity or expression are necessary. In contrast to previous studies (Hu & Wilson, 1997; Mangia et al. 2003) lactate dips during the early phase of neuronal activation were not observed. These discrepancies can always be attributed to divergent stimulus protocols. However, microdialysis measurements report only bulk changes in extracellular metabolites with limited temporal and spatial resolution, and rapid and localized metabolic events may be averaged out. Given the cellular and metabolic heterogeneity of nervous tissue on the micrometer scale (Fig. 1), measurements that directly resolve cellular responses (Kasischke et al. 2004) are ultimately required. A closer look at the cerebellar cortex may explain why: astrocytes and neurons are the principal structural elements in the molecular layer (Fig. 1A). Their processes are intimately associated in a convoluted, sometimes parallel spatial arrangement, implying that the distances for trans-cellular diffusion and transport between adjacent processes are appreciably shorter than the distances within a single cell (Fig. 1B). Astrocytic glutamate transporters are strongly expressed in the cerebellar cortex and they are perfectly positioned as a coupling site for neuron–glia interactions. In fact, astrocytic glutamate update sites ensheath Purkinje cells so tightly that their somas are delineated by a continuous EAAT2 band (Fig. 1C). The cellular distribution of cytochrome oxidase in the cerebellar cortex (Fig. 1D and E) implies that neuronal elements are profoundly oxidative, while the Bergmann glia exhibit considerably lower oxidative capacities, indirectly supporting the Pellerin & Magistretti model. Figure 1 Glutamate transporters and oxidative capacities in the rat cerebellar cortex The study by Caesar et al. suggests an unexpected pathway for lactate production in the CNS and diverts our attention to AMPA-mediated events in Purkinje cells and Bergmann glia. It will be exciting to see whether future studies can directly identify the cellular origin of extracellular lactate and its ultimate fate.