Fasting duration impacts ribosome protein 6 phosphorylation in zebrafish brain: New insights in aquatic organisms' welfare.

BACKGROUND: Short- or mid-term fasting, full or partial, triggers metabolic response known to have in turn health effects in an organism. At central level, the metabolic stimulus triggered by fasting is known to be perceived firstly by hypothalamic neurons. In the field of neuroscience, ribosomal protein S6 (S6) phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signalling activation or as a marker for neuronal activity. The aim of this study is addressed to evaluate whether the phosphorylation of S6 occurs in the central neurons of zebrafish exposed to four (short-term) and seven (mid-term) days of complete fasting. METHODS: Group-housed adult zebrafish were exposed to four and seven days of complete food withdrawal. At the end of the experimental period, Western blotting analyses were carried out to measure the expression levels of the phosphorylated S6 (pS6) by comparing the two experimental conditions versus the control group. The same antibody was then used to identify the distribution pattern of pS6 immunoreactive neurons in the whole brain and in the taste buds. RESULTS: We did not observe increased pS6 levels expression in the brain of animals exposed to short-term fasting compared to the control, whereas the expression increased in brain homogenates of animals exposed to mid-term fasting. pS6 immunoreactivity was reported in some hypothalamic neurons, as well as in the dorsal area of telencephalon and preoptic area, a neurosecretory region homolog to the mammalian paraventricular nucleus. Remarkably, we observed pS6 immunostaining in the sensory cells of taste buds lining the oral epithelium. CONCLUSIONS: Taken together, our data show that in zebrafish, differently from other fish species, seven days of fasting triggers neuronal activity. Furthermore, the immunostaining on sensory cells of taste buds suggests that metabolic changes may modulate also peripheral sensory cells. This event may have valuable implications when using zebrafish to design metabolic studies involving fasting as well as practical consequences on the animal welfare, in particularly stressful conditions, such as transportation.

Rapid innervation and physiological epidermal regeneration by bioengineered dermis implanted in mouse.

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

Early endocannabinoid-mediated depolarization-induced suppression of excitation delays the appearance of the epileptic phenotype in synapsin II knockout mice.

The mechanism underlying the transition from the pre-symptomatic to the symptomatic state is a crucial aspect of epileptogenesis. SYN2 is a member of a multigene family of synaptic vesicle phosphoproteins playing a fundamental role in controlling neurotransmitter release. Human SYN2 gene mutations are associated with epilepsy and autism spectrum disorder. Mice knocked out for synapsin II (SynII KO) are prone to epileptic seizures that appear after 2 months of age. However, the involvement of the endocannabinoid system, known to regulate seizure development and propagation, in the modulation of the excitatory/inhibitory balance in the epileptic hippocampal network of SynII KO mice has not been explored. In this study, we investigated the impact of endocannabinoids on glutamatergic and GABAergic synapses at hippocampal dentate gyrus granule cells in young pre-symptomatic (1-2 months old) and adult symptomatic (5-8 months old) SynII KO mice. We observed an increase in endocannabinoid-mediated depolarization-induced suppression of excitation in young SynII KO mice, compared to age-matched wild-type controls. In contrast, the endocannabinoid-mediated depolarization-induced suppression of inhibition remained unchanged in SynII KO mice at both ages. This selective alteration of excitatory synaptic transmission was accompanied by changes in hippocampal endocannabinoid levels and cannabinoid receptor type 1 distribution among glutamatergic and GABAergic synaptic terminals contacting the granule cells of the dentate gyrus. Finally, inhibition of type-1 cannabinoid receptors in young pre-symptomatic SynII KO mice induced seizures during a tail suspension test. Our results suggest that endocannabinoids contribute to maintaining network stability in a genetic mouse model of human epilepsy.

Investigating the Effect of Surface Hydrophilicity on the Destiny of PLGA-Poloxamer Nanoparticles in an In Vivo Animal Model.

This study aimed to examine the impact of different surface properties of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (P NPs) and PLGA-Poloxamer nanoparticles (PP NPs) on their in vivo biodistribution. For this purpose, NPs were formulated via nanoprecipitation and loaded with diphenylhexatriene (DPH), a fluorescent dye. The obtained NPs underwent comprehensive characterization, encompassing their morphology, technological attributes, DPH release rate, and thermodynamic properties. The produced NPs were then administered to wild-type mice via intraperitoneal injection, and, at scheduled time intervals, the animals were euthanized. Blood samples, as well as the liver, lungs, and kidneys, were extracted for histological examination and biodistribution analysis. The findings of this investigation revealed that the presence of poloxamers led to smaller NP sizes and induced partial crystallinity in the NPs. The biodistribution and histological results from in vivo experiments evidenced that both, P and PP NPs, exhibited comparable concentrations in the bloodstream, while P NPs could not be detected in the other organs examined. Conversely, PP NPs were primarily sequestered by the lungs and, to a lesser extent, by the kidneys. Future research endeavors will focus on investigating the behavior of drug-loaded NPs in pathological animal models.

Effects of Olea europaea L. Polyphenols on the Animal Welfare and Milk Quality in Dairy Cows.

Here, we evaluated the effect of dietary supplementation with an Olea europaea L. extract on the animal welfare and milk quality of dairy cows. Thirty Italian Holstein-Friesian dairy cows in the mid-lactation phase (90 to 210 days) were blocked into experimental groups based on parity class (namely, primiparous (P) (n = 10), secondiparous (S) (n = 10) and pluriparous (PL) (n = 10)) and received, for 60 days, Phenofeed Dry® at 500 mg/cow/day. Milk and blood samples were collected before the start of the treatment (T0), subsequently every 15 days (T1-T4) and at 45 days after the end of treatment (T5). In the serum, glucose and triglycerides, stress, the thyroid, lactation and sex hormones were measured; in the milk, lysozyme content as well as the fatty acid profile were assessed. In the whole animal, the enriched feed helped to maintain hormonal parameters in the physiological range while producing hypoglycemic (T4 vs. T0, for P and PL p < 0.001) and hypolipidemic effects (T4 vs. T0, for P p < 0.001 and for PL p < 0.01). At the milk level, it resulted in a reduction in total fat (T5 vs. T0, for P, S and PL p < 0.001) and in the saturated fatty acids (SFAs)/monounsaturated fatty acids (MUFAs) ratio paralleled by an increase in polyunsaturated fatty acids (PUFAs) (T5 vs. T0, for P, S and PL p < 0.001), protein content (lysozyme (T4 vs. T0, for P and PL p < 0.001)) and lactose (T5 vs. T0, for P, S and PL p < 0.001). Thus, the inclusion of natural bioactive molecules such as O. europaea L. polyphenols in the dairy cow diet may help to improve animal welfare and milk quality.

Data repurposing from digital home cage monitoring enlightens new perspectives on mouse motor behaviour and reduction principle.

In this longitudinal study we compare between and within-strain variation in the home-cage spatial preference of three widely used and commercially available mice strains-C57BL/6NCrl, BALB/cAnNCrl and CRL:CD1(ICR)-starting from the first hour post cage-change until the next cage-change, for three consecutive intervals, to further profile the circadian home-cage behavioural phenotypes. Cage-change can be a stressful moment in the life of laboratory mice, since animals are disturbed during the sleeping hours and must then rapidly re-adapt to a pristine environment, leading to disruptions in normal motor patterns. The novelty of this study resides in characterizing new strain-specific biological phenomena, such as activity along the cage walls and frontality, using the vast data reserves generated by previous experimental data, thus introducing the potential and exploring the applicability of data repurposing to enhance Reduction principle when running in vivo studies. Our results, entirely obtained without the use of new animals, demonstrate that also when referring to space preference within the cage, C57BL/6NCrl has a high variability in the behavioural phenotypes from pre-puberty until early adulthood compared to BALB/cAnNCrl, which is confirmed to be socially disaggregated, and CRL:CD1(ICR) which is conversely highly active and socially aggregated. Our data also suggest that a strain-oriented approach is needed when defining frequency of cage-change as well as maximum allowed animal density, which should be revised, ideally under the EU regulatory framework as well, according to the physiological peculiarities of the strains, and always avoiding the "one size fits all" approach.

Integration of micro-CT and histology data for vasculature morpho-functional analysis in tissue regeneration.

The demand for artificial or bioartificial engineered tissues is increasing today in regenerative medicine techniques to replace and restore the physiological function of damaged tissues. Such engineered constructs hold different properties depending on the tissue to be replicated. As for vascularized tissues, complex biocompatible structures, namely scaffolds, play a key role in supporting oxygen and nutrient supply, thus sustaining tissue neoformation and integration with the host. Scaffold architecture significantly impacts its regenerative potential, while preclinical trials are essential to define scaffold-host interactions. In compliance with the 3 R principle, there is a clear need to optimize both the procedures to evaluate scaffold performance and the analysis methodology decreasing the number of animals required to gain consistent data. In parallel, current technologies used in preclinical research generate huge amounts of data that need to be elaborated and interpreted correctly. Therefore, we designed this study to evaluate the results of scaffold integration with the host tissue after implantation in a mouse subcutaneous pocket model. We evaluated the angiogenic response developed by the host and the degree of scaffold integration by using a combined morphometric approach based on both histological and micro-CT analyses. Six-layer scaffolds, made of polycaprolactone (PCL) microspheres, with an ordered structure were produced by thermal sintering. Scaffolds were then implanted in BALB/c mice and retrieved 21 days post-implantation when the animals were deeply anesthetized and perfused with Microfil, a contrast agent for micro-CT. Here, we describe a method to extract quantitative data from micro-CT reconstructions such as (i) total vessel volume; (ii)% of vessel penetration; (iii) distribution of vessel diameters. The general principle of this approach is the refinement of the region of interest (ROI), thus producing a volume of interest (VOI) that matches scaffold volume. This VOI serves as a dataset from which to extract volumetric information. Then VOIs are divided into three identical parts, proximal, median, and distal, to follow the vessel progression into the scaffold, thus obtaining their depth of penetration (DoP). By this methodology, we observed in mean, among the analyzed samples, a vessel invasion for 1,38 mm3 corresponding to the 1,53% of the scaffold volume. We then looked at the diameter distribution being this value a key indicator of vessel maturity, highlighting that 55% of vessels fall into the range from 5,99-53.99 µm while the remaining 45% are distributed into intervals from 54 to 136 µm. In parallel, to evaluate tissue integration in detail, histological and immunofluorescent analyses were performed to look at vessel distribution and collagen synthesis. Histological results strongly correlate with the micro-CT data providing, however, an overview of the ingrowth tissues. In addition, by immunofluorescent analysis we demonstrate that newly formed vessels are mature at the considered time point and tissue collagen deposition is widespread within the scaffolds. Collectively, we propose a new method to track vessel formation by using a multi-modal approach posing the basis for: i) the fabrication of novel scaffolds for Tissue Engineering; ii) the integration of detailed information for a wide range of morphological and functional analyses.

Positive association between plasmatic levels of orexin A and the endocannabinoid-derived 2-arachidonoyl lysophosphatidic acid in Alzheimer's disease.

A regular sleep-wake cycle plays a positive function that preserves synaptic plasticity and brain activity from neuropathological injuries. The hypothalamic neuropeptide orexin-A (OX-A) is central in sleep-wake regulation and has been found to be over-expressed in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) suffering from sleep disturbances. OX-A promotes the biosynthesis of 2-arachidonoylglycerol (2-AG), which, in turn, could be phosphorylated to 2-arachidonoyl lysophosphatidic acid (2-AGP). The reorganization of the actin cytoskeleton during neurite retraction is one of the best-characterized effects of lysophosphatidic acids. However, less information is available regarding the reorganization of the neuronal microtubule network in response to OX-A-induced 2-AG and, possibly consequent, 2-AGP production in AD patients. This is of special relevance also considering that higher 2-AG levels are reported in the CSF of AD patients. Here, we found a positive correlation between OX-A and 2-AGP concentrations in the plasma, and an increase of 2-AGP levels in the CSF of AD patients. Furthermore, a negative correlation between the plasmatic 2-AGP levels and the mini-mental state examination score is also revealed in AD patients. By moving from the human patients to in vitro and in vivo models of AD we investigated the molecular pathway linking OX-A, 2-AG and 2-AGP to the phosphorylation of pT231-Tau, which is a specific early plasma biomarker of this disorder. By LC-MS analysis we show that OX-A, via OX-1R, induces 2-AG biosynthesis via DAGLα, and in turn 2-AG is converted to 2-AGP in primary hippocampal neurons. By confocal microscopy and western blotting assay we found an OX-A- or 2-AGP-mediated phosphorylation of Tau at threonine 231 residue, in a manner prevented by LPA1R (2-AGP receptor) or OX1R (OX-A receptor) antagonism with AM095 or SB334867, respectively. Finally, by patch-clamp recording we documented that 2-AGP-mediated pT231-Tau phosphorylation impairs glutamatergic transmission in the mouse hippocampus. Although further additional research is still required to clarify the potential role of orexin signaling in neurodegeneration, this study provides evidence that counteraction of aberrant OX-A signaling, also via LPA-1R antagonism, may be beneficial in the mild-to-moderate age-related cognitive decline associated with sleep disturbances.

Effect of microneedles shape on skin penetration and transdermal drug administration.

Microneedle (MN) patches are highly efficient and versatile tools for transdermal drug administration, in particular for pain-free, self-medication and rapid local applications. Diffraction ultraviolet (UV) light lithography offers an advanced method in fabricating poly(ethylene glycol)-based MNs with different shapes, by changing both the UV-light exposure time and photomask design. The exposure time interval is limited at obtaining conical structures with aspect ratio < 1:3, otherwise MNs exhibit reduced fracture load and poor indentation ability, not suitable for practical application. Therefore, this work is focused on a systematic analysis of the MN's base shapes effects on the structural characteristics, skin penetration and drug delivery. Analyzing four different base shapes (circle, triangle, square and star), it has been found that the number of vertices in the polygon base heavily affects these properties. The star-like MNs reveal the most efficient skin penetration ability (equal to 40 % of -their length), due to the edges action on the skin during the perforation. Furthermore, the quantification of the drug delivered by the MNs through ex-vivo porcine skin shows that the amounts of small molecules released over 24 h by star-like MNs coated by local anesthetic (Lidocaine) and an anti-inflammatory (Diclofenac epolamine) drugs are 1.5× and 2× higher than the circular-MNs, respectively.

Immunolocalization of Nesfatin-1 in the Gastrointestinal Tract of the Common Bottlenose Dolphin Tursiops truncatus.

First identified as an anorexigenic peptide, in the last decades, several studies have suggested that Nesfatin-1 (Nesf-1) is a pleiotropic hormone implicated in numerous regulatory processes in peripheral organs and tissues. In vertebrates, Nesf-1 is indeed expressed in the central nervous system and peripheral organs. In this study, we characterized the pattern of Nesf-1 distribution within the digestive tract of the common bottlenose dolphin (Tursiops truncatus), composed of three gastric chambers and an intestine without a clear subdivision in the small and large intestine, also lacking a caecum. Our results indicated that Nesf-1 is widely distributed in cells of the mucosal epithelium of the gastric chambers. Most of the immunoreactivity was observed in the second chamber, compared to the first and third chambers. Immunopositivity was also found in nerve fibers and neurons, scattered or/and clustered in ganglion structures along all the examined gastrointestinal tracts. These observations add new data on the highly conserved role of Nesf-1 in the mammalian digestive system.

An Alkaloid from a Highly Invasive Seaweed Increases the Voracity and Reproductive Output of a Model Fish Species.

The invasive macroalga Caulerpa cylindracea has spread widely in the Mediterranean Sea, becoming a favorite food item for native fish for reasons yet unknown. By using a combination of behavioral, morphological, and molecular approaches, herein we provide evidence that the bisindole alkaloid caulerpin, a major secondary metabolite of C. cylindracea, significantly increases food intake in the model fish Danio rerio, influencing the regulation of genes involved in the orexigenic pathway. In addition, we found that the compound improves fish reproductive performance by affecting the hypothalamus-pituitary-gonadal axis. The obtained results pave the way for the possible valorization of C. cylindracea as a sustainable source of a functional feed additive of interest to face critical challenges both in aquaculture and in human nutrition.

Central and Peripheral NPY Age-Related Regulation: A Comparative Analysis in Fish Translational Models.

NPY is among the most abundant neuropeptides in vertebrate brain and is primarily involved in the regulation of food intake. The NPY system is also associated with the aging process showing beneficial effects on neuronal survival via autophagy modulation. Here, we explore the age-related regulation of NPY in the brain and foregut of the shortest- and longest-lived fish species, Nothobranchius furzeri and Danio rerio, respectively. These two research models, despite some similarities, display profound biological differences making them attractive vertebrates to elucidate the mechanisms underlying the regulation of neuropeptide synthesis and function. It is noteworthy that in both fish species only Npya has been identified, while in the other teleosts two classes of NPY (Npya and Npyb) have been annotated. Our findings document that in both species: (i) NPY is centrally regulated; (ii) NPY levels increase in the brain during aging; (iii) NPY is localized in the enteroendocrine cells as well as in the myenteric plexus and drastically decreases in old animals. According to our data, the age-related regulation in the gut resembles that described in other vertebrate species while the increased levels in the brain offer the unique possibility to explore the role of NPY in model organisms to develop future experimental and translatable approaches.

Puberty onset curve in CD (Sprague Dawley) and Long Evans outbred male rats.

The exact timing of puberty is fundamental in preclinical studies. In male rats, the age at sexual maturity varies considerably between 40 and 60 days of age. Here, we summarize pubertal onset evaluation of two outbred rat strains (Crl:CD(SD) and Crl:LE), relying on the balano-preputial separation test. Evaluation was carried out on animals under standard barrier conditions, from four to nine weeks of age. In the Crl:CD(SD) population, 90% of males gained puberty at week 6, and 100% in the following weeks, whereas 75% of Crl:LE reached puberty at week 6, 90% at week 7 and 100% from week 8. Remarkably, in both strains, puberty onset was gained at the average weight of 200 g, suggesting that weight range, not only age range, can be considered a biomarker of puberty onset in these two strains. On the contrary, descended testes cannot be considered an additional factor to identify full puberty onset either in Crl:CD(SD) or Crl:LE rats.

Neuronal Phenotype of col4a1 and col25a1: An Intriguing Hypothesis in Vertebrates Brain Aging.

Collagens are the most abundant proteins in vertebrates and constitute the major components of the extracellular matrix. Collagens play an important and multifaceted role in the development and functioning of the nervous system and undergo structural remodeling and quantitative modifications during aging. Here, we investigated the age-dependent regulation of col4a1 and col25a1 in the brain of the short-lived vertebrate Nothobranchius furzeri, a powerful model organism for aging research due to its natural fast-aging process and further characterized typical hallmarks of brain aging in this species. We showed that col4a1 and col25a1 are relatively well conserved during vertebrate evolution, and their expression significantly increases in the brain of N. furzeri upon aging. Noteworthy, we report that both col4a1 and col25a1 are expressed in cells with a neuronal phenotype, unlike what has already been documented in mammalian brain, in which only col25a1 is considered a neuronal marker, whereas col4a1 seems to be expressed only in endothelial cells. Overall, our findings encourage further investigation on the role of col4a1 and col25a1 in the biology of the vertebrate brain as well as the onset of aging and neurodegenerative diseases.

Orexin-A and endocannabinoids are involved in obesity-associated alteration of hippocampal neurogenesis, plasticity, and episodic memory in mice.

The mammalian brain stores and distinguishes among episodic memories, i.e. memories formed during the personal experience, through a mechanism of pattern separation computed in the hippocampal dentate gyrus. Decision-making for food-related behaviors, such as the choice and intake of food, might be affected in obese subjects by alterations in the retrieval of episodic memories. Adult neurogenesis in the dentate gyrus regulates the pattern separation. Several molecular factors affect adult neurogenesis and exert a critical role in the development and plasticity of newborn neurons. Orexin-A/hypocretin-1 and downstream endocannabinoid 2-arachidonoylglycerol signaling are altered in obese mice. Here, we show that excessive orexin-A/2-arachidonoylglycerol/cannabinoid receptor type-1 signaling leads to the dysfunction of adult hippocampal neurogenesis and the subsequent inhibition of plasticity and impairment of pattern separation. By inhibiting orexin-A action at orexin-1 receptors we rescued both plasticity and pattern separation impairment in obese mice, thus providing a molecular and functional mechanism to explain alterations in episodic memory in obesity.

Neurotrophins in the Brain of Teleost Fish: The State of the Art.

Neurotrophins are evolutionary well-conserved molecules, and fish constitute valuable vertebrate models to explore their pleiotropic role in the brain. In addition to an introduction on the evolutionary importance of using fish in biomedicine and their neuroanatomy in comparison with mammals, here we review the available literature on the molecular evolution of neurotrophins and their receptors in teleost fish as well as their role in the fish brain, from the early stages of development until adulthood and aging. Among neurotrophins, BDNF is the most well studied in the brain of teleost fish, and we report data on the functional involvement of the BDNF/TrkB system in the development of the visual system and in the mechanisms of adult brain regeneration. With the exception of neuroanatomical expression, much less is known about the role of the other members of neurotrophin family in fish brain. We hope that this chapter opens new avenues leading to a better understanding of the complex and multifaceted roles of neurotrophins in the brain of fish and other vertebrates.

Phenotyping spontaneous locomotor activity in inbred and outbred mouse strains by using Digital Ventilated Cages.

Mouse strains differ markedly in all behaviors, independently of their genetic background. We undertook this study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in the study design to ensure experimental reproducibility and comply with essential animal welfare principles.

A combined morphometric approach to feature mouse kidney vasculature.

Physiological kidney function is closely related to the state of the vascular network. Disorders, such as capillary rarefaction, predispose to chronic kidney disease (CKD). In this context, deepening of the methodologies for studying the renal vascular network can be of basic importance. To meet this need, numerous animal models and, in parallel, several methods have been developed. In this work we propose a protocol to accurately feature kidney vasculature in mouse, however, the same protocol is suitable to be applied also to other animal models. The approach is multiparametric and mainly based on micro-computed tomography (µCT) technique. Micro-ct allows to study in detail the vascular network of any organ by exploiting the possibility to perfuse the sample with a contrast agent. The proposed protocol provides a fast and reliable method to extract quantitative information from the µCT scan by using only the basic functions of the software supplied by the scanner without any additional analysis. Through iterative cropping of the scanned ROI and calculation of a sample-specific threshold we calculated that the average volume of a female BALB/c kidney of eighth weeks is 147.8 mm3 (5.4%). We also pointed out that the average volume of the vascular network is 4.9% (0.3%). In parallel we performed traditional histological and immunofluorescence techniques to integrate the information gained via µCT and to frame them in the tissue context. Vessel count on histological sections showed a different density in the different regions of the organ parenchyma, in detail, vessel density in the cortex was 19.03 ± 2.51 vessels/ROI while in the medulla it was 10.6 ± 1.7 vessels/ROI and 5.4 ± 1.3 vessels/ROI in the outer and inner medulla, respectively. We then studied vessel distribution in the renal parenchyma which showed that the 55% of vascular component is included in the cortex, the 30% in the outer medulla and the 15% in the inner medulla. Collectively, we propose an integrated approach that can be particularly useful in the preclinical setting to characterize the vasculature of any organ accurately and rapidly.

Distinct Pattern of NPY in Gastro-Entero-Pancreatic System of Goat Kids Fed with a New Standardized Red Orange and Lemon Extract (RLE).

The use of natural compounds as feed additive is also increasing in farm animals, thanks to the beneficial effect on both animals and consumers health. Here, we questioned whether natural extracts, such as red orange and lemon extract (RLE) rich in flavanones, anthocyanins, and other polyphenols, used as feed additives could display an effect on the neuropeptide Y (NPY) in the gastro-entero-pancreatic tract of goat kids. NPY is one of the most abundant neuropeptides in mammals, known for its orexigenic role although it is involved in many central and peripheral functions. We carried out immunohistochemical analyses on samples of abomasum, duodenum and pancreas collected from two experimental groups: one fed with standard diet and one with standard diet + RLE. For the first time we document NPY distribution in the abomasum, duodenum and pancreas of goats and observe the highest number of NPY positive cells in neuroendocrine cells of duodenum. Remarkably, upon RLE feed supplementation, NPY immunoreactive cells increased significantly in abomasal epithelium and pancreatic islets but not in duodenum, likely due to pH variation of abomasum and duodenum. Our observations represent a baseline for future studies on the interaction between neuropeptides and polyphenols, used as feed additive.