Towards optogenetic laryngeal pacemaking - first proof-of-concept study in pigs

• Applicants: Professor Dr. Dirk Beutner; Professor Dr. Tobias Brügmann; Dr.-Ing. Patrick Ruther
• Subject Area: Anatomy and Physiology ; Otolaryngology, Phoniatrics and Audiology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 452139556

Bilateral paralysis of the Nervus laryngeus recurrens leads to fixed vocal cords and impedes air passage through the larynx. As a consequence, patients suffer from acute dyspnea which can currently only be treated with destructive surgical interventions. Alternatively, electrical stimulation of the intralaryngeal muscles has been explored in animals and humans since the 1970s. This technique is able to open the vocal cords. Unfortunately, the co-stimulation of antagonistic muscles and pain nerves, as well as other technical issues hinder its use in clinical routine.The working hypothesis underlying this application is that optogenetic stimulation of specific muscle groups should enable pain-free laryngeal pacemaking and restore laryngeal function in patients. We have recently established optogenetic stimulation of intact skeletal muscles taking advantage of transgenic mice expressing the light-gated non-selective cation channel Channelrhodopsin2 (ChR2). Single light pulses were found to induce twitch contractions and repetitive illumination tetanic contractions in soleus muscles. In addition, we could demonstrate in explanted larynges selective stimulation of distinct intralaryngeal muscle groups for separate vocal cord opening as well as their closing. Herein, we want to explore optogenetic laryngeal pacemaking by testing gene transfer of ChR2 and light stimulation in pigs in vivo. This large animal model allows to probe the translational potential of optogenetic stimulation due to the similarities of larynx anatomy and physiology between pigs and humans. To express ChR2 in the intralaryngeal muscles, we will inject adeno-associated viruses (AAV) locally into the postlaryngeal region. First, we will identify the amount of AAV required for the sufficient expression of ChR2 and light stimulation by injecting 4 different dosages of AAV and testing the efficacy after two weeks. Second; we will focus on determining the long-term efficacy by performing the analysis eight weeks after the injection with the best dosage from the first experiments. During all time points, we will explore the potential immune response against the gene transfer and ChR2. Finally, we will test ex vivo and in vivo a already developped implantable light device which can be used in the future in conscious animals. Towards these aims, we have already established all surgical interventions on dead pigs and a set of various experiments to characterize and quantify the potential immune response. Thus, the long-term goal of this project is the establishment and assessment of optogenetic pacemaking of the larynx to restore vocal cord mobility in cases of laryngeal paralysis. These experiments of optogenetic stimulation are the first in a representative large animal model outside the immune-privileged eye and brain, and can thus serve as model for many other translational applications of optogenetics.

• DFG Programme: Research Grants