Multiphoton Microscopy and Fluorescence Lifetime Imaging of Hypometabolism in Epileptic Tissue

Michael J. Levene, Ph.D.

Yale University School of Medicine

Biomedical Engineering
Funded in December, 2008: $100000 for 1 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY .

LAY SUMMARY

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Using Cellular Imaging to Understand Dysfunction of Brain Metabolism in Epilepsy

Yale researchers will use cellular imaging techniques in brain tissues of epilepsy patients, which have been surgically removed to treat medication-resistant seizures, to determine the cellular cause of slowed brain metabolism in epilepsy.

Epilepsy seizures occur when neurons in large areas of brain tissue fire synchronously.  Paradoxically, while epilepsy is characterized by brain cell hyperactivity during a seizure, a consistent feature of epilepsy is a reduced metabolic rate, or “hypometabolism.”  Research has focused on neurons that use the excitatory neurotransmitter glutamate to communicate with one another, and on cells called “astrocytes” that ordinarily clear away excess glutamate from the spaces (called synapses) where one neuron passes glutamate on to the next.  Metabolic problems in the affected neurons might be involved.  Or, if astrocytes fail to fully clear away glutamate, neurons may become overstimulated simultaneously and produce a seizure.  The investigators hypothesize that malfunctioning astrocytes have defects in their mitochondria, the metabolic energy-producing engine of cells.

With initial Dana support, the Yale researchers demonstrated the feasibility of using multiphoton fluorescence microscopy in combination with fluorescence lifetime imaging to study mitochondria in astrocytes in brain tissues in an epilepsy animal model.  Multiphoton imaging measures exactly how long it takes, after stimulating a molecule (called NADH) that is involved in energy metabolism in mitochondria, to emit fluorescence.  Fluorescence lifetime imaging removes artifacts affecting interpretation of these results. 

Now the investigators will use these imaging techniques in surgically removed brain tissue from epilepsy patients, to study NADH molecules in the mitochrondria of neurons and astrocytes.  They expect to determine that astrocytic energetics are impaired, as determined by abnormal NADH lifetime distributions, while the surviving neurons are relatively normal in this regard.

ABSTRACT

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Multiphoton Microscopy and Fluorescence Lifetime Imaging of Hypometabolism in Epileptic Tissue

Hypometabolism is a consistent feature of human epileptic tissue as described by PET and SPECT imaging.  This is paradoxical given that this tissue is able to generate and support energetically demanding seizures.  The underlying mechanism(s) for this observation are not known, but histological and isotopic data from excised epileptic tissue suggests damaged mitochondria may play a role.  A limitation of these studies is that it has not been possible to spatially resolve whether the impaired mitochondria are neuronal and/or glial, and to what degree each cell population contributes to the overall reduction in metabolism.  The development of imaging tools for assessing metabolic function with the spatial resolution sufficient to discriminate neuronal and glial populations is critical to elucidating the underlying source of pathology. 

Multiphoton microscopy (MPM) of intrinsic fluorescence from NADH has both the spatial resolution and sensitivity to discriminate between these cell populations.  Because NADH is autofluorescent, but NAD+ is not, changes in NADH fluorescence can be correlated with changes in the redox state of NADH, which is altered at several stages of both oxidative and non-oxidative metabolism.  In addition, the techniques of fluorescence lifetime imaging (FLIM) and fluorescence polarization anisotropy decays can yield further information on the distribution of NADH.  As NADH binds to various enzymes, changes to both fluorescence and anisotropy lifetimes can be measured and their respective amplitudes correspond to distributions of NADH binding among different enzymes and freely diffusing NADH.

We propose to use MPM, FLIM and anisotropy lifetime imaging to support the hypothesis that the hypometabolism seen in epileptic tissue reflects impaired mitochondrial function in abnormal (reactive) astrocytes.  We will use tissue slices prepared from epileptic human hippocampus or from an animal model of the pathology, the chronically epileptic, pilocarpine-treated mouse.  Taken together, FLIM and anisotropy images will reveal both the number of mitochondria and give several indications of any alterations in mitochondrial function.  The spatial resolution of MPM is sufficient to identify mitochondria within cells and to reveal changes to their number in pathological tissue.  FLIM and anisotropy can reveal changes in NADH concentration and binding in response to chemical stimulation that may be altered in epileptic tissue.  Anisotropy can additionally reveal changes to mitochondrial and somatic viscosity associated with swelling, another indicator of dysfunction.  These studies will expand our understanding of neural metabolism in a common form of human epilepsy and have the potential to drive the development of technology that can better identify epileptogenic tissue.

INVESTIGATOR BIOGRAPHIES

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Michael J. Levene, Ph.D.

Prof. Levene received his B.S. in Optics from the University of Rochester in 1992, and his Ph.D. from the Computation and Neural Systems Program at The California Institute of Technology in 1998.  His Pd.D. thesis, entitled “Optical Neural Networks,” included the invention of Shift Multiplexing for holographic data storage, which was later licensed to Holoplex, Inc.  Prof. Levene then went on to work as a post-doctoral research associate in Biophysics under Watt W. Webb at Cornell University, where he developed zero-mode waveguides for single molecule spectroscopy at physiologically relevant concentrations and for single-molecule DNA sequencing.  This technology has been licensed to, and is the foundation for, Pacific Biosciences, Inc.  While at Cornell, he also was the first to demonstrate in vivo multiphoton imaging with gradient index lenses for accessing deep structures within the mouse brain.

Prof. Levene’s current research program at Yale continues to expand the use of micro-optics for in vivo multiphoton microscopy (MPM), including applications to image-guided neurosurgery, and also involves application of MPM and fluorescence lifetime imaging to investigate the underlying nature of metabolic abnormalities in epilepsy.  Indeed, his lab is one of the only labs in the world performing MPM on human brain tissue on a regular basis.  Prof. Levene is also expanding the range of application of single molecule biophysical techniques, such as fluorescence fluctuation spectroscopy, to clinical laboratory diagnostics.