MULTIMODAL ASSESSMENT OF CETACEAN CENTRAL NERVOUS AUDITORY PATHWAYS WITH EMPHASIS ON FORENSIC DIAGNOSTICS OF ACOUSTIC TRAUMA

Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment.