RESUMEN
Imaging of living animals allows the study of metabolic processes in relation to cellular structures or larger functional entities. To enable in vivo imaging during long-term time-lapses in planarians, we combined and optimized existing protocols, resulting in an easily reproducible and inexpensive procedure. Immobilization with low-melting-point agarose eliminates the use of anesthetics, avoids interfering with the animal during imaging-functionally or physically-and allows recovering the organisms after the imaging procedure. As an example, we used the immobilization workflow to image the highly dynamic and fast-changing reactive oxygen species (ROS) in living animals. These reactive signaling molecules can only be studied in vivo and mapping their location and dynamics during different physiological conditions is crucial to understand their role in developmental processes and regeneration. In the current protocol, we describe both the immobilization and ROS detection procedure. We used the intensity of the signals together with pharmacological inhibitors to validate the signal specificity and to distinguish it from the autofluorescent nature of the planarian.
Asunto(s)
Planarias , Animales , Planarias/fisiología , Especies Reactivas de Oxígeno/metabolismo , Diagnóstico por ImagenRESUMEN
A strict coordination between pro- and antioxidative molecules is needed for normal animal physiology, although their exact function and dynamics during regeneration and development remains largely unknown. Via in vivo imaging, we were able to locate and discriminate between reactive oxygen species (ROS) in real-time during different physiological stages of the highly regenerative planarian Schmidtea mediterranea. All ROS signals were strong enough to overcome the detected autofluorescence. Combined with an in situ characterisation and quantification of the transcription of several antioxidant genes, our data showed that the planarian gut and epidermis have a well-equipped redox system. Pharmacological inhibition or RNA interference of either side of the redox balance resulted in alterations in the regeneration process, characterised by decreased blastema sizes and delayed neurodevelopment, thereby affecting tails more than heads. Focusing on glutathione, a central component in the redox balance, we found that it is highly present in planarians and that a significant reduction in glutathione content led to regenerative failure with tissue lesions, characterised by underlying stem cell alterations. This exploratory study indicates that ROS and antioxidants are tightly intertwined and should be studied as a whole to fully comprehend the function of the redox balance in animal physiology.