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Introduction: The auditory system of dolphins and whales allows them to dive in dark waters, hunt for prey well below the limit of solar light absorption, and to communicate with their conspecific. These complex behaviors require specific and sufficient functional circuitry in the neocortex, and vicarious learning capacities. Dolphins are also precocious animals that can hold their breath and swim within minutes after birth. However, diving and hunting behaviors are likely not innate and need to be learned. Our hypothesis is that the organization of the auditory cortex of dolphins grows and mature not only in the early phases of life, but also in adults and aging individuals. These changes may be subtle and involve sub-populations of cells specificall linked to some circuits., Methods: In the primary auditory cortex of 11 bottlenose dolphins belonging to three age groups (calves, adults, and old animals), neuronal cell shapes were analyzed separately and by cortical layer using custom computer vision and multivariate statistical analysis, to determine potential minute morphological differences across these age groups., Results: The results show definite changes in interneurons, characterized by round and ellipsoid shapes predominantly located in upper cortical layers. Notably, neonates interneurons exhibited a pattern of being closer together and smaller, developing into a more dispersed and diverse set of shapes in adulthood., Discussion: This trend persisted in older animals, suggesting a continuous development of connections throughout the life of these marine animals. Our findings further support the proposition that thalamic input reach upper layers in cetaceans, at least within a cortical area critical for their survival. Moreover, our results indicate the likelihood of changes in cell populations occurring in adult animals, prompting the need for characterization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Graïc, Corain, Finos, Vadori, Grisan, Gerussi, Orekhova, Centelleghe, Cozzi and Peruffo.)

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cetartiodactyls include terrestrial and marine species, all generally endowed with a comparatively lateral position of their eyes and a relatively limited binocular field of vision. To this day, our understanding of the visual system in mammals beyond the few studied animal models remains limited. In the present study, we examined the primary visual cortex of Cetartiodactyls that live on land (sheep, Père David deer, giraffe); in the sea (bottlenose dolphin, Risso's dolphin, long-finned pilot whale, Cuvier's beaked whale, sperm whale and fin whale); or in an amphibious environment (hippopotamus). We also sampled and studied the visual cortex of the horse (a closely related perissodactyl) and two primates (chimpanzee and pig-tailed macaque) for comparison. Our histochemical and immunohistochemical results indicate that the visual cortex of Cetartiodactyls is characterized by a peculiar organization, structure, and complexity of the cortical column. We noted a general lesser lamination compared to simians, with diminished density, and an apparent simplification of the intra- and extra-columnar connections. The presence and distribution of calcium-binding proteins indicated a notable absence of parvalbumin in water species and a strong reduction of layer 4, usually enlarged in the striated cortex, seemingly replaced by a more diffuse distribution in neighboring layers. Consequently, thalamo-cortical inputs are apparently directed to the higher layers of the column. Computer analyses and statistical evaluation of the data confirmed the results and indicated a substantial correlation between eye placement and cortical structure, with a markedly segregated pattern in cetaceans compared to other mammals. Furthermore, cetacean species showed several types of cortical lamination which may reflect differences in function, possibly related to depth of foraging and consequent progressive disappearance of light, and increased importance of echolocation., (© 2021. The Author(s).)

Background: Cornea is a composite tissue exhibiting nonlinear and time-dependent mechanical properties. Corneal ulcers are one of the main pathologies that affect this tissue, disrupting its structural integrity and leading to impaired functions. In this study, uniaxial tensile and stress-relaxation tests are developed to evaluate stress-strain and time-dependent mechanical behaviour of porcine corneas., Results: The samples are split in two groups: some corneas are analysed in an unaltered state (healthy samples), while others are injured with alkaline solution to create an experimental ulcer (lesioned samples). Furthermore, within each group, corneas are examined in two conditions: few hours after the enucleation (fresh samples) or after 7 days in a specific culture medium for the tissue (cultured samples). Finally, another condition is added: corneas from all the groups undergo or not a cross-linking treatment. In both stress-strain and stress-relaxation tests, a weakening of the tissue is observed due to the imposed conditions (lesion, culture and treatment), represented by a lower stiffness and increased stress-relaxation., Conclusions: Alkali-induced corneal stromal melting determines changes in the mechanical response that can be related to a damage at microstructural level. The results of the present study represent the basis for the investigation of traditional and innovative corneal therapies., (© 2021. The Author(s).)

The lungs of cetaceans undergo anatomical and physiological adaptations that facilitate extended breath-holding during dives. Here, we present new insights on the ontogeny of the microscopic anatomy of the terminal portion of the airways of the lungs in five cetacean species: the fin whale (Balaenoptera physalus); the sperm whale (Physeter macrocephalus), the Cuvier's beaked whale (Ziphius cavirostris); the bottlenose dolphin (Tursiops truncatus); and the striped dolphin (Stenella coeruleoalba). We (a) studied the histology of the terminal portion of the airways; (b) used immunohistochemistry (IHC) to characterize the muscle fibers with antibodies against smooth muscle (sm-) actin, sm-myosin, and desmin; (c) the innervation of myoelastic sphincters (MESs) with an antibody against neurofilament protein; and (d) defined the diameter of the terminal bronchioles, the diameter and length of the alveoli, the thickness of the septa, the major and minor axis, perimeter and section area of the cartilaginous rings by quantitative morphometric analyses in partially inflated lung tissue. As already reported in the literature, in bottlenose and striped dolphins, a system of MESs was observed in the terminal bronchioles. Immunohistochemistry confirmed the presence of smooth muscle in the terminal bronchioles, alveolar ducts, and alveolar septa in all the examined species. Some neurofilaments were observed close to the MESs in both bottlenose and striped dolphins. In fin, sperm, and Cuvier's beaked whales, we noted a layer of longitudinal smooth muscle going from the terminal bronchioles to the alveolar sacs. The morphometric analysis allowed to quantify the structural differences among cetacean species by ranking them into groups according to the adjusted mean values of the morphometric parameters measured. Our results contribute to the current understanding of the anatomy of the terminal airways of the cetacean lung and the role of the smooth muscle in the alveolar collapse reflex, crucial for prolonged breath-holding diving., (© 2020 Wiley Periodicals LLC.)

In Paralympic sports, the goal of functional classifications is to minimize the impact of impairment on the outcome of the competition. The present cross-sectional study aimed to investigate aerobic and anaerobic personalized tests in Paralympic athletes and to correlate them with the classification of the international wheelchair rugby federation (IWRF). Sixteen elite players of the Italian wheelchair rugby team volunteered for the study. Aerobic (incremental test to exhaustion) and anaerobic (Wingate 30s all-out test, 5 and 10-meter sprint test, shuttle test, isometric test) sport-performance measurements were correlated singularly or grouped (Z scores) with the classification point. Moreover, a multivariate permutation-based ranking analysis investigated possible differences in the overall level of performance among the adjacent classified groups of players, considering the scores of each test. A statistically significant correlation between the performance parameters and the IWRF functional classification considering both aerobic and anaerobic personalized tests was detected (0.58 ≤ r ≤ 0.88; 0.0260 ≤ p ≤ 0.0001). The multivariate permutation-based ranking analysis showed differences only for the low-pointers versus mid-pointers ( p = 0.0195) and high-pointers ( p = 0.0075). Although single performance parameters correlated with athletes' classification point, results of the multivariate permutation-based ranking analysis seem to suggest considering only the most significant anaerobic and sport-specific performance parameters among athletes. These should be combined with the physical assessment and the qualitative observation, which are already part of the classification process to improve its effectiveness.

The evolution of the brain in apes and man followed a joint pathway stemming from common ancestors 5-10 million years ago. However, although apparently sharing similar organization and neurochemical properties, association areas of the isocortex remain one of the cornerstones of what sets humans aside from other primates. Brodmann's area 44, the area of Broca, is known for its implication in speech, and thus indirectly is a key mark of human uniqueness. This latero-caudal part of the frontal lobe shows a marked functional asymmetry in humans, and takes part in other complex functions, including learning and imitation, tool use, music and contains the mirror neuron system (MNS). Since the main features in the cytoarchitecture of Broca's area remains relatively constant in hominids, including in our closest relative, the chimpanzee Pan troglodytes , investigations on the finer structure, cellular organization, connectivity and eventual asymmetry of area 44 have a direct bearing on the understanding of the neural mechanisms at the base of our language. The semi-automated image analysis technology that we employed in the current study showed that the structure of the cortical layers of the chimpanzee contains elements of asymmetry that are discussed in relation to the corresponding human areas and the putative resulting disparity of function., (Copyright © 2020 Graïc, Peruffo, Corain, Centelleghe, Granato, Zanellato and Cozzi.)

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated ( p < 0.00) and healthy ( p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference ( p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations.

The laminar organization of the motor cortex of the sheep and other large domestic herbivores received scarce attention and is generally considered homologous to that of rodents and primates. Thickness of the cortex, subdivision into layers and organization are scarcely known. In the present study, we applied different modern morphological, mathematical and image-analyses techniques to the study of the motor area that controls movements of the forelimb in the sheep. The thickness of the cortex resulted comparable to that of other terrestrial Cetartiodactyls (but thicker than in marine Cetartiodactyls of similar body mass). The laminar organization showed marked development of layer 1, virtual absence of layer 4, and image analysis suggested prevalence of large irregular neural cells in the deeper layers. Diffusion tensor imaging revealed robust projections from the motor cortex to the pyramids in the brainstem, and well evident tracts descending to the tegmentum of the mesencephalon and dorsal pons. Our data contrast the general representation of the motor system of this species, considered to be predominantly based on extra-pyramidal tracts that originate from central pattern generators in the brainstem.