How does atmospheric pressure cold helium plasma affect the biomechanical behaviour on alkali-lesioned corneas?

BACKGROUND: Melting corneal ulcers are a serious condition that affects a great number of animals and people around the world and it is characterised by a progressive weakening of the tissue leading to possible severe ophthalmic complications, such as visual impairment or blindness. This disease is routinely treated with medical therapy and keratoplasty, and recently also with alternative regenerative therapies, such as cross-linking, amniotic membrane transplant, and laser. Plasma medicine is another recent example of regenerative treatment that showed promising results in reducing the microbial load of corneal tissue together with maintaining its cellular vitality. Since the effect of helium plasma application on corneal mechanical viscoelasticity has not yet been investigated, the aim of this study is first to evaluate it on ex vivo porcine corneas for different exposition times and then to compare the results with previous data on cross-linking treatment. RESULTS: 94 ex vivo porcine corneas divided into 16 populations (healthy or injured, fresh or cultured and treated or not with plasma or cross-linking) were analysed. For each population, a biomechanical analysis was performed by uniaxial stress-relaxation tests, and a statistical analysis was carried out considering the characteristic mechanical parameters. In terms of equilibrium normalised stress, no statistically significant difference resulted when the healthy corneas were compared with lesioned plasma-treated ones, independently of treatment time, contrary to what was obtained about the cross-linking treated corneas which exhibited more intense relaxation phenomena. CONCLUSIONS: In this study, the influence of the Helium plasma treatment was observed on the viscoelasticity of porcine corneas ex vivo, by restoring in lesioned tissue a degree of relaxation similar to the one of the native tissue, even after only 2 min of application. Therefore, the obtained results suggest that plasma treatment is a promising new regenerative ophthalmic therapy for melting corneal ulcers, laying the groundwork for further studies to correlate the mechanical findings with corneal histology and ultrastructural anatomy after plasma treatment.

Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

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.

Mechanical behaviour of healthy versus alkali-lesioned corneas by a porcine organ culture model.

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.

Sexually Diergic Trophic Effects of Estradiol Exposure on Developing Bovine Cerebellar Granule Cells.

In the mammalian brain, the differentiation of neural cells and the developmental organization of the underlying circuitry are influenced by steroid hormones. The estrogen 17-ß estradiol (E2) is one of the most potent regulators of neural growth during prenatal life, synthetized locally from steroid precursors including prenatal testicular testosterone. Estradiol promotes brain differentiation counting sexually dimorphic neural circuits by binding to the estrogen receptors, ER-α and ER-ß. The cerebellum has been described as a site of estrogen action and a potentially sexually dimorphic area. The goal of this study was to analyze the capacity of E2 to affect the growth of male and female fetal bovine cerebellar granule. We performed primary cultures of fetal cerebellar granules, and verified the mRNA expression of the ER-α and ER-ß in both sexes. Moreover, the distribution of ERs in the male and female cerebellar granules of the second fetal stage was characterized by immunohistochemistry. We measured morphological parameters in presence (or absence) of estradiol administration, focusing on the variations of the dendritic branching pattern of granule neurons. By using the nonparametric combination and permutation testing approach, we proposed a sophisticated multivariate statistical analysis to demonstrate that E2 induces multifarious and dimorphic changes in the granule cells. E2 exerts trophic effects in both female and male granules and this effect is stronger in female. Male granules treated with E2 became similar to female control granule. Bos taurus species has a long gestation and a large brain that offers an interesting alternative in comparative neuroscience.

Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles.

Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier's beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-ß. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier's beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment.

The prefrontal cortex of the bottlenose dolphin (Tursiops truncatus Montagu, 1821): a tractography study and comparison with the human.

Cetaceans are well known for their remarkable cognitive abilities including self-recognition, sound imitation and decision making. In other mammals, the prefrontal cortex (PFC) takes a key role in such cognitive feats. In cetaceans, however, a PFC could up to now not be discerned based on its usual topography. Classical in vivo methods like tract tracing are legally not possible to perform in Cetacea, leaving diffusion-weighted imaging (DWI) as the most viable alternative. This is the first investigation focussed on the identification of the cetacean PFC homologue. In our study, we applied the constrained spherical deconvolution (CSD) algorithm on 3 T DWI scans of three formalin-fixed brains of bottlenose dolphins (Tursiops truncatus) and compared the obtained results to human brains, using the same methodology. We first identified fibres related to the medio-dorsal thalamic nuclei (MD) and then seeded the obtained putative PFC in the dolphin as well as the known PFC in humans. Our results outlined the dolphin PFC in areas not previously studied, in the cranio-lateral, ectolateral and opercular gyri, and furthermore demonstrated a similar connectivity pattern between the human and dolphin PFC. The antero-lateral rotation of the PFC, like in other areas, might be the result of the telescoping process which occurred in these animals during evolution.

Age-related changes in the primary auditory cortex of newborn, adults and aging bottlenose dolphins (Tursiops truncatus) are located in the upper cortical layers.

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.

Estradiol effects on intracellular Ca(2+) homeostasis in bovine brain-derived endothelial cells.

Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.

Evidence of melatonin secretion in cetaceans: plasma concentration and extrapineal HIOMT-like presence in the bottlenose dolphin Tursiops truncatus.

The pineal gland is generally believed to be absent in cetaceans, although few and subsequently unconfirmed reports described the organ in some species. The recent description of a complete and photographed pineal body in a bottlenose dolphin (Tursiops truncatus) prompted us to examine a series of 29 brains of the same species, but no gland was found. We then decided to investigate if the main product of the gland, melatonin, was nevertheless produced and present in the plasma of this species. We collected plasma and serum samples from a series of captive bottlenose dolphins for a period of 7 months spanning from winter to summer and we determined the indoleamine concentration by radio-immunoassay (RIA). The results demonstrated for the first time a quantitative assessment of melatonin production in the blood of a cetacean. Melatonin levels were comparable to those of terrestrial mammals (5.15-27.74 pg/ml daylight concentration), with indications of both seasonal and daily variation although the presence of a circadian rhythm remains uncertain. Immunohistochemical analyses using as a marker hydroxyindole-O-methyl-transferase (HIOMT, the key enzyme involved in the biosynthesis of the hormone), suggested extrapineal melatonin production by the retina, the Harderian gland and the gut. The enzyme was unequivocally localized in all the three tissues, and, specifically, ganglion cells in the retina showed a very strong HIOMT-immunoreactivity. Our results suggest that further research might reveal unexplored aspects of melatonin production in cetaceans and deserves special attention and further efforts.

The orbitofrontal cortex of the sheep. Topography, organization, neurochemistry, digital tensor imaging and comparison with the chimpanzee and human.

Areas dedicated to higher brain functions such as the orbitofrontal cortex (OFC) are thought to be unique to hominidae. The OFC is involved in social behavior, reward and punishment encoding and emotional control. Here, we focused on the putative corresponding area in the sheep to assess its homology to the OFC in humans. We used classical histology in five sheep (Ovis aries) and four chimpanzees (Pan troglodytes) as a six-layered-cortex primate, and Diffusion Tensor Imaging (DTI) in three sheep and five human brains. Nissl's staining exhibited a certain alteration in cortical lamination since no layer IV was found in the sheep. A reduction of the total cortical thickness was also evident together with a reduction of the prevalence of layer one and an increased layer two on the total thickness. Tractography of the sheep OFC, on the other hand, revealed similarities both with human tracts and those described in the literature, as well as a higher number of cortico-cortical fibers connecting the OFC with the visual areas in the right hemisphere. Our results evidenced the presence of the basic components necessary for complex abstract thought in the sheep and a pronounced laterality, often associated with greater efficiency of a certain function, suggested an evolutionary adaptation of this prey species.

The primary visual cortex of Cetartiodactyls: organization, cytoarchitectonics and comparison with perissodactyls and primates.

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.

Aromatase expression in cultured fetal sheep astrocytes after nitrosative/oxidative damage.

Aromatase, the enzyme converting androgens into estrogens, is involved in many brain processes such as neural differentiation and plasticity or the prevention of cell death. We have previously observed an increase in aromatase immunoreactivity in sheep neurons exposed in vitro to the oxidant 3-nitro-L: -tyrosine. However, little is known regarding the way that sheep astrocytes cope with nitrosative stress, a condition occurring in sheep in the pathogenesis of neurodegenerative disorders such as scrapie and Maedi-Visna. Our aim has been to evaluate the effects of 3-nitro-L-tyrosine on astrocyte primary cultures from 90-day-old fetal sheep brain. Living cells were observed and characterized by immunofluorescence with a GFAP antibody, which indicated that the majority of the cells were astrocytes. A viability assay was performed on both untreated and treated cells. Reverse transcription with the polymerase chain reaction was undertaken to monitor time- and dose-dependent variations in aromatase gene expression. Stressed astrocytes showed signs of deterioration, were reduced in number, and appeared round with few short processes; the cell death rate was ∼30%. Aromatase expression was detected starting from a 24-h exposure to 1 mM 3-nitro-L-tyrosine and reached the highest levels at 72 h. Thus, oxidative damage probably results in the local production of neuroprotective estradiol by reactive astrocytes via the aromatization of testosterone.

Topographical and structural characterization of the V1-V2 transition zone in the visual cortex of the long-finned pilot whale Globicephala melas (Traill, 1809).

The visual system of cetaceans is at best poorly understood. With a handful of electrophysiological studies and a limited number of histological preparations from well-preserved specimen, the investigation of the principles underlying the cortical organization in cetaceans remains a challenge. In the course of our current investigation, we identified the transition from V2 to V1 in the long-finned pilot whale Globicephala melas, only recognizable through immunocytochemistry, and a similar if not homologue transition in the sheep Ovis aries. Our results emphasize the importance of differential pattern recognition in which the application of different markers uncovers a diversity in a delphinid's cortex, formerly widely considered as uniform and archetypal. In fact, the evidence that we present suggests the existence of relatively unacknowledged areas beyond the well-known sensory territories in cetaceans.

Microscopic anatomical, immunohistochemical, and morphometric characterization of the terminal airways of the lung in cetaceans.

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.

Neurochemical characterization of the hypothalamus of the early fetal and newborn alpaca Vicugna pacos.

In this study we performed a neurochemical characterization of the hypothalamus in the developing alpaca (Vicugna pacos) with the aim of revealing the distributions of immunoreactive (-ir) cells containing parvalbumin (PV), calbindin (CB), calretinin (CR), the somatostatin (SOM), the enzyme aromatase P450 (P450Arom), the estrogen receptor α (ER-α), and estrogen receptor ß (ER-ß) in embryonal stages, early fetal age, and in the newborn. This analysis has been carried out on embryos at 20, 30, 45 days, fetuses at 90 days, and newborn alpaca. Our immunohistochemical results revealed no cells-ir throughout the embryonic hypothalami of 20, 30, and 45 days. On the fetal stage of 90 days, SOM-ir cells were observed in the lateral hypothalamus and the ventromedial nuclei of the tuberal region. We checked for the presence of P450Arom-ir cells in the periventricular area and dorsomedial hypothalamic nucleus of the tuberal region. In these fetal stages, no PV-ir, CB-ir, CR-ir or ERs-ir cells were identified. In the newborn, the PV-ir, CB-ir, CR-ir, and SOM-ir cells were detected in both the anterior and tuberal hypothalamic area. The P450Arom-ir cells the ER-α-ir and ER-ß-ir cells were found in the anterior hypothalamus. Our results offer a contribution in the future purpose to obtain a time-expression pattern of the considered markers in alpaca during gestation and represents a foundation for future investigations on the alpaca brain to define the cross talk between PV, CB, CR, P450Arom, SOM, and ERs in the hypothalamus, the strategic region for the control of the reproductive behavior.

Asymmetry in the Cytoarchitecture of the Area 44 Homolog of the Brain of the Chimpanzee Pan troglodytes.

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.

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

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.

Mapping of the Early Intrauterine Morphogenesis in the Alpaca (Vicugna pacos): External Features and Development of the Cephalic Vesicle in Comparison with the Progressive Carnegie Scale.

In this study, we characterized the morphological aspects of the early development of the head of the alpaca (Vicugna pacos) and identified the main structures of the central nervous system during the first trimester of pregnancy. The topography and the cytoarchitecture of the fetal brain regions were described by histological analysis of the brain sections. We performed this analysis on alpaca embryos and fetuses presumably aged 20, 30, 45, and 90 days. For the description of the external body structures we considered the shape of the head, the development of the optic primordium, the dorsal curvature of the body, the limb buds, the umbilical cord and relative vessels, and the thickness and transparency of the skin. The prosencephalic, mesencephalic, and the rhomboencephalic vesicles were described by analyzing sagittal sections of the head. The present article provides the first progressive morphological and anatomical description of alpaca brain during early development. A detailed study represents an important basis to further understand the phases of prenatal development in this species, since information about alpaca embryology in incomplete and reproductive failure is a relevant factor. These data are important also for interspecies comparisons and application of reproductive biotechnologies. Anat Rec, 302:1226-1237, 2019. © 2018 Wiley Periodicals, Inc.

The motor cortex of the sheep: laminar organization, projections and diffusion tensor imaging of the intracranial pyramidal and extrapyramidal tracts.

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.

Accumulation of copper and other metal ions, and metallothionein I/II expression in the bovine brain as a function of aging.

Accumulation of metal ions in the brain contributes to heighten oxidative stress and neuronal damage as evidenced in aging and neurodegenerative diseases, both in humans and in animals. In the present paper we report the analysis of Cu, Zn and Mn in the brain of two series of respectively young (8-16 months) and adult (9-12 years) bovines. Our data indicate that the concentrations of Cu varied of one order of magnitude between 1.67 and 15.7microg/g wet tissue; the levels of Zn varied between 6.13 and 17.07microg/g wet tissue and the values of Mn resulted between 0.19 and 1.24microg/g wet tissue. We found relevant age-dependent differences in the distribution of Cu and Zn, whose concentrations were markedly higher in older animals. By contrast, Mn seemed to redistribute in the different cerebral areas rather than drastically change with age. Tissues from bovine brain were also analysed immunohistochemically for the presence and distribution of metallothionein I/II and also for the expression of glial fibrillary acidic protein. Metallothionein I/II immunoreactive elements included ependymal cells lining the lateral ventricles and neural cells in middle layer of the cerebellar cortex. No age differences were evident between calves and adult. The presence of liquor-contacting metallothionein I/II in cells confirms that their functions in the central nervous system are not yet completely established.