Multiphoton Microscopy and Fluorescence Lifetime Imaging of Hypometabloism in Epileptic Tissue

Michael Levene, Ph.D.

Yale University

Funded in December, 2006: $100000 for 1 years
LAY SUMMARY . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Using Cellular Imaging to Understand Dysfunction of Brain Metabolism in Epilepsy

Yale researchers will test the feasibility of using multiphoton fluorescence imaging in animal model tissue of epilepsy to determine whether this technique can reveal how brain metabolism is slowed in this disease and how this process contributes to seizure development.

Research has demonstrated that brain metabolism is slowed in people with epilepsy, but the reasons for this hypometabolism, and its consequences for seizure development, are not currently understood.  Neurons that communicate with one another through release of the excitatory neurotransmitter glutamate depend upon astrocytes to clear away excess glutamate from the synapses that connect neurons to one another. If glutamate is insufficiently cleared, neurons may become overstimulated, leading to seizures.  The investigators hypothesize that malfunctioning astrocytes have defects in their mitochondria, a component that ordinarily produces energy for nerve cells. 

They will investigate this hypothesis using multiphoton fluorescence microscopy to observe the functioning of astrocytes and neurons in animal model epilepsy tissue.  This imaging technique can measure exactly how long it takes, after stimulating a molecule (called NADH) that is involved in energy metabolism in mitochondria, to emit fluorescence. Through this imaging technique, they will determine whether there are too few NADH molecules, or whether the levels are normal but the molecules are malfunctioning.

Significance:  If the researchers demonstrate the feasibility of using this imaging technique to identify the basis of metabolic dysfunction in astrocytes, the technique then can be tested in tissue removed from patients undergoing surgery for intractable epilepsy.  Ultimately, the findings may lead to development of drugs that specifically target metabolic dysfunction of astrocytes.  Moreover, this imaging technique could be used by neurosurgeons prior to surgery for intractable epilepsy, as a marker to more accurately differentiate normal from epileptic brain tissue.

HYPOTHESIS

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Hypothesis:
Hypometabolism is a consistent feature of human epileptic tissue as described by PET and SPECT imaging.  NADH, a ubiquitous coenzyme, is an intrinsic fluorophore that provides cellular metabolic information.  We investigate metabolic pathologies in human and rodent epilepsy models using multiphoton microscopy of NADH.  We hypothesize the use of multiphoton microscopy-based tools will reveal that hypometabolism seen in epileptic tissue reflects impaired mitochondrial function in either abnormal (reactive) astrocytes and/or neurons.

Goals:
We will use tissue slices prepared from epileptic human hippocampus and slices from the rat model of epilepsy (kainate-treated).  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 multiphoton microscopy 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.

Methods:
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 of NADH intrinsic fluorescence has both the spatial resolution and sensitivity to discriminate between these cell populations. Because NADH is fluorescent, 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.

SELECTED PUBLICATIONS

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Levene M.J., Dombeck D.A., Kasischke K.A., Molloy R.P., and Webb W.W., In vivo multiphoton microscopy of deep brain tissue. J Neurophysiol. 2004 Apr;91(4):1908-12.