Project Grant R21DC021730

CHARACTERIZATION OF THE COCHLEAR AQUEDUCT - THIS PROPOSAL WILL DETERMINE THE MORPHOLOGICAL, MOLECULAR, AND FUNCTIONAL MECHANISMS ON HOW THE COCHLEAR AQUEDUCT (CA) CHANGES OVER THE LIFESPAN. THIS WILL BE THE FIRST HISTOLOGICAL AND MOLECULAR CHARACTERIZATION OF THE COCHLEAR AQUEDUCT OVER THE LIFESPAN OF CBA/CAJ MICE. AIM 1: A COMPLETE CHARACTERIZATION OF PASSAGE OF TRACERS, SPHERES, OR IMMUNE CELLS THROUGH THE CA WITH INCREASING AGE IS LACKING AND THEREFORE THIS INFORMATION IS NEEDED FOR THE FUTURE DEVELOPMENT OF USING THE COCHLEAR AQUEDUCT AS A POINT OF ENTRY TO THE COCHLEA FOR RESCUE STRATEGIES FOR THE INNER EAR. THE ROUTE USED TO DELIVER THESE SUBSTANCES WILL BE THROUGH THE CA BY INJECTING INTO THE CSF IN THE CISTERNAE MAGNA. EXPERIMENTS ARE DESIGNED TO DETERMINE THE RELATIVE DISTRIBUTION OF TRACERS THROUGHOUT THE COCHLEA BY USING GADOVIST AND CONTRAST-ENHANCED MR IMAGING AT DIFFERENT AGES. THE HIGHEST RESOLUTION OF PRE-CLINICAL MRI (A 9.4 T BRUKER MACHINE) WILL BE USED FOR COCHLEAR FLUID IMAGING IN LIVING ANIMALS. THE STUDY WILL DETERMINE THE SIZE LIMITATIONS OF PASSAGE THROUGH THE BARRIER MEMBRANE (LOCATED AT THE COCHLEAR END OF THE AQUEDUCT) AND IF IT CHANGES WITH INCREASING AGE. TO DETERMINE THE ACCESSIBILITY OF MICROSPHERES (0.2-2MM), WHICH IS A NEW TECHNOLOGY THAT CAN DELIVER DRUGS, STEM CELLS, AND OTHER THERAPEUTICS, HISTOLOGICAL INVESTIGATIONS WILL BE PERFORMED AFTER THEY ARE INJECTED INTO THE CISTERNA MAGNUM AND THEN HISTOLOGICALLY QUANTIFIED. WE HAVE FOUND MACROPHAGES AND LYMPHOID MARKERS IN THE CA AND WE HYPOTHESIZE THAT THE CA COULD BE A SOURCE OF IMMUNE CELLS THAT ENTER THE COCHLEA. WE WILL INJECT GFP-LABELLED CX3CR1 MONOCYTES ISOLATED FROM CX3CR1CREER-EYFP/WT MICE AVAILABLE IN OUR FACILITY, INTO THE CISTERNAE MAGNUM BEFORE OR AFTER A PTS-INDUCING NOISE EXPOSURE (110 DB, 2H, 6-12 KHZ IN AWAKE ANIMALS) KNOWN TO TRIGGER INFLAMMATORY PROCESSES IN THE COCHLEA AND EVALUATE BY HISTOLOGY WHETHER THESE CELLS DELIVERED IN THE CSF HAVE BEEN POPULATING THE COCHLEA IN GREATER ABUNDANCE THAN IN SHAM EXPOSED ANIMALS. AIM 2: A COMPLETE CHARACTERIZATION OF THE CELLULAR IDENTITY OF THE TISSUE SURROUNDING THE CA IS NEEDED TO BETTER UNDERSTAND ITS ROLE AS A COCHLEAR GATE KEEPER. LASER CAPTURE MICRODISSECTION COUPLED WITH NEXT GENERATION SEQUENCING (LCM- SMART- SEQ3) DEVELOPED AT THE KAROLINSKA INSTITUTE AND WILL DETERMINE THE MOLECULAR COMPOSITION AND THE CELLULAR IDENTIFY OF THE INNER TISSUE LAYER. THIS UNIQUE WORK WILL ESTABLISH A MORPHOLOGICAL, PHYSIOLOGICAL, AND MOLECULAR CHARACTERIZATION OF THE CA. KNOWLEDGE WILL BE GENERATED REGARDING HOW THE INFLUX OF FLUIDS FROM THE CA AQUEDUCT IS REGULATED DURING THE LIFESPAN. THE RESULTS FROM THIS PROJECT WILL ESTABLISH A FUNDAMENTAL BASIS FOR UNDERSTANDING THE CA AS A PASSAGE ROUTE TO AND FROM THE COCHLEA AND ITS USE FOR PRE-CLINICAL DELIVERY STRATEGIES TO TREAT COCHLEAR DISORDERS.