Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons.

Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.