ANR TECSAN - VSD-IR Project 2012-2015

The use of voltage-sensitive dyes imaging as a molecular voltmeter contrast agent offers the possibility to explore the cerebral cortex activity in real time with excellent spatial and temporal resolutions. However, despite the remarkable development and progress in the quality and sensitivity of the dyes, this technology is not suitable for in vivo studies without brain surgery. Since the beginning of 2011, we are involved with SANOFI R&D in an ambitious and multidisciplinary project, which primary goal is to develop new voltage sensitive probes for in vivo real time imaging of cortical activity in a non invasive way.

 

Since almost a century electrical activities of hearth and brain have been visualized by electrocardiography (ECG) or electroencephalogry (EEG). Since three decades, these electrical activities of hearth and brain could also be visualized by fluorescent dyes. The general idea is to change the electrical signal of a cell into an optical signal. For that purpose fluorescent molecules are introduced in the plasma membrane of cells. These fluorescent dyes are lipophilic and localized in the lipidic bilayers of the plasma membrane of the cells.  The fluorescent properties of the dyes will be modified with the changes of membrane voltage. These fluorescent dyes sensitive to potential are known under the name of “Voltage Sensitive Dyes” or VSDs. The goal of the present proposal is to develop new VSDs and to use them for visualization of hearth and brain electrical activities in several animal models including non-human primates. The final goal is to develop new non-toxic VSDs which could be use in humans. The first application would be done in the neurosurgery of epileptic foyer with the aim to precisely localize the pathological tissue.

 

VSDs have already been used with great success in hearth and brain imaging. Electrical maps of the complete intact tissue are obtained. VSDs could also been used to monitor electrical activity of a single neuron and even to follow the action potential in a single axon. Electrophysiological techniques and the optical techniques using VSDs have the same high temporal resolution in the millisecond. The spatial resolution of VSDs is much more superior to classical EEG or ECG recordings. The cellular resolution is achieved with VSDs.

The recent progresses of functional imaging are amazing. VSDs are widely used in animal models by cardiologists and neuroscientists. On the other hand, VSDs are not widely used in the clinics as they are not exempted from toxic effect. One of the first goal of our project is the preparation of VSDs devoid of toxic effect.

The fluorescent excitation emission spectra of the present available VSDs are in the visible. Visible light is absorbed in the tissues and to get enough fluorescent signals, illumination has to be high enough. Strong illumination causes phototoxicity. Preparation of infrared fluorescent compounds should decrease tissue absorption and phototoxicity.  One of the main tasks of this proposal is centred on the chemical synthesis of new VSDs with fluorescent properties in the infrared. Already three classes of new VSDs with fluorescent properties in the near infrared have already been synthetized. Several of the fluorescent molecules developed are excited at over 700 nm with emission over 800 nm. This choice of excitation-emission is optimal for use in living blood perfused tissue. The maximum absorption of haemoglobin is between 433 nm and 577 nm. Over 850 nm, water becomes a problem.

 

The final goal of this proposal is to prepare new VSDs for in vivo functional imaging of electrical activity in humans for cardiology and neurology. Already in the last two years more than ten fluorescent dyes have been synthetized. They belong to three different families. They will be tested in vitro on neuronal cultures in order to select the dyes giving a good electrochromicity i.e. the relation between changes in cellular potential and fluorescent signals. The selected dyes will then be tested in vivo. In cardiology they will be tested in Langendorff-perfused rat and pig hearths and compared with dyes currently used. The selected dyes will also be tested for imaging neocortical activities in response to sensory stimulation in mice and also in non-human primates. If one the selected dyes has passed toxicity and regulatory tests, it will be used in neurosurgical treatment of epileptic seizures. It is expected that the topical application of the VSD on the exposed cortex could help the neurosurgeon in delineating the dysplasic tissue from the healthy one.

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