An in vitro model for assessment of excitotoxicity induced by glutamate and neuroprotection using digitized fluorescent imaging of mitochondrial potentials

  • Mohammed Sherwan Muhyaddin College of Pharmacy, Hawler Medical University, Erbil, Iraq
Keywords: neuronal culture, digitized florescent imaging, glutamic acid, neurotoxicity


Background and objectives: The established methods to demonstrate the neuroprotective efficacy of drugs still use in vivo animal tests. The objective of the present study, therefore, was to develop in vitro screening systems based on digitized fluorescent imaging of individual neurons from neuronal cell cultures as a practical model for assessment of neuroprotectors during excitotoxicity.

Methods: Cortical cells from embryonic mice were cultivated on glass coverslips Cells were loaded with the fluorescence dye rhodamine-123. The coverslips were mounted in a temperature controlled flow-through chamber. The superfusion chamber was mounted on an inverted microscope. The emission light after excitation of the dye was measured with an intensified CCD camera, accordingly the changes in the mitochondrial membrane potential were monitored.

Results: Neurotoxic concentrations of glutamic acid induced a rapid and irreversible damage to mitochondrial membrane potential. The non-competitive NMDA-receptor antagonist MK-801 (dizocilpine) preserved neuronal viability. The loss of rhodamine-123 fluorescence highly correlated with the ongoing neuronal cell death and was shown to be a suitable parameter to determine the neuroprotective action of pharmaceutical compounds.

Conclusion: Loss of the mitochondrial membrane potential can be used as dynamic markers of cellular injury in vitro. Presumably, the in vivo animal experiments are required and cannot be avoided completely. However, the described approach can at least enlighten the preliminary neuroprotective effect of drugs or their critical concentrations directly on the neuronal level and by that way avoid a large number of animal tests.


Sattler R and Tymianski M. Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Mol Neurobiol , 2001, 24: 107-129.

.Blandini F., Porter R. H., Greenamyre J, T, Glutamate and Parkinson’s disease. Molecular Neurobiology 1996, 12, 73-94

Hynd, M. R., Scott, H. L., and Dodd, P. R. Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease. Neurochem. Int. 2004, 45, 583–595.

Lynch, D. R. and Guttmann, R, P. Excitotoxicity: Perspectives Based on N-Methyl-D-Aspartate Receptor Subtypes, J. Pharmacol. Exp. Ther., 2002; 300: 717

Chavez J. C., Hurko O, Barone F C, and Feuerstein G Z, Pharmacologic interventions for stroke: looking beyond the thrombolysis time window into the penumbra with biomarkers, not a stopwatch. Stroke; 2009, 40: 558 - 563.

McAuley M.A., Rodent models of focal ischemia, Cerebrovascular and Brain Metabolism Reviews, 7, 152-180, 1995

Fox G., Gallacher D., Shevde S., Loftus J.and Swayne G., Anatomic variation of the middle cerebral artery in the Sprague-Dawley rat, Stroke, 1993, 24, 2087-2093

Oberpichler-Schwenk H. and Krieglstein J., Primary cultures of neurons for testing neuroprotective drug effects, Journal of Neural Transmission, [Suppl] , 1994, 44, 1-20.

,R.J. and Reynolds, I.J., Mitochondrial depolarization in glutamate stimulated neurons: an early signal specific to excitotoxin exposure, Journal of Neuroscience, 1996, 16, 5688-5697.

Koh J.Y. and Choi D.W Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay, Journal of Neuroscience Methods,1987 20, 83-90.

Brookes P. S., Yoon Y., Robotham J. L., Anders M. W., and Sheu S., Calcium, ATP, and ROS: a mitochondrial love-hate triangle Am J Physiol Cell Physiol 2004, 287, C817–C833

Duchen M. R., Mitochondria and calcium: from cell signalling to cell death J. Physiol. 2000, 529, 57-68

Rother J., Neuroprotection does not work! Stroke, 2008, 39, 523–524.

Faden A. I. and Stoica B., Neuroprotection: Challenges and Opportunities, Arch Neurol., 2007, 64, 794-800

How to Cite
Muhyaddin, M. (2018). An in vitro model for assessment of excitotoxicity induced by glutamate and neuroprotection using digitized fluorescent imaging of mitochondrial potentials. Zanco Journal of Medical Sciences (Zanco J Med Sci), 16(1), 9-15.
Original Articles