Enterobacteriaceae and Salmonella contamination of wild boar (Sus scrofa) carcasses: comparison between different sampling strategies.

During 2020, a total of 64 wild boar carcasses were tested for Enterobacteriaceae count (EBC), Salmonella and Yersinia enterocolitica in the abdominal region (i) within 5 h after hunting in the game collection point and (ii) before dressing and processing in the game-handling establishment (GHE) (49 carcasses-average time interval between (i) and (ii): 4.3 days). Because of COVID-19 restrictions, 15 carcasses were transported to a near slaughterhouse (average time interval between (i) and (ii): 2.3 days). Mesenteric lymph nodes (MLNs) were collected and tested for Salmonella and Y. enterocolitica. Results are shown in relation to sampling A (49 carcasses-GHE) and sampling B (15 carcasses-slaughterhouse). Sampling A: EBC median values were (i) 2.51 log10 CFU/cm2 and (ii) 2.79 log10 CFU/cm2. EBC increase between (i) and (ii) was statistically significant (p = 0.001). Salmonella prevalence on carcasses varied from (i) 2.0 to (ii) 6.1%. Sampling B: EBC median values were (i) 3.1 log10 CFU/cm2 and (ii) 3.32 log10 CFU/cm2. EBC increase between (i) and (ii) was not statistically significant (p = 0.191). Salmonella prevalence on carcasses varied from (i) 6.7 to (ii) 0.0%. The prevalence (sampling A + B) of lymphatic Salmonella carriers was 7.8% (5/64). From carcasses and/or MNLs, the serovars Enteritidis, Typhimurium, Agama, Zaiman and Diarizonae O:50 (z) were detected. Y. enterocolitica was never isolated. Long chilling periods prior to wild game processing should be avoided, and carcasses should be tested at GHE rather than after shooting to proper reflect the microbial load of wild boar meat entering the food chain.

Modeling pulmonary fibrosis through bleomycin delivered by osmotic minipump: a new histomorphometric method of evaluation.

The systemic delivery of bleomycin (BLM) to mice through subcutaneously implanted osmotic minipumps may be used to experimentally mimic the typical features of systemic sclerosis and related interstitial lung diseases. The published studies on this model principally have focused on induced dermal modifications, probably because lung lesions are typically mild, subpleurally localized, and difficult to analyze. The use of high BLM doses to increase their severity has been proposed but is ethically questionable because of the compromising of animal welfare. We propose a tailored histomorphometric method suitable to detect and quantify this type of mild lung lesions. Using a two-step automated image analysis, a peripheral region of interest with a depth of 250 µm from the pleural edge was defined on whole slide images, and the fibrotic foci were histomorphometrically characterized. The effects of different BLM doses on lung alterations were evaluated in C57BL/6 mice and 60 U/kg resulted in a fair compromise between fibrotic lesions and animal welfare. This dose was also tested in time course experiments. The analysis revealed a peak of histological fibrotic-like alterations, cytokine expression, metalloprotease, and macrophagic activation between the 21st and 28th day after pump implant. The induced dermal fibrosis was characterized by the progressive loss of the white dermal adipose layer, an increase in dermal thickness, dermal hyperplasia, and more compacted collagen fibers. Despite the trend toward spontaneous resolution, our model allowed a double organ readout of the BLM effect and the identification of a therapeutic window for testing pharmacological compounds without using life-threatening doses.

Profile of gamma-delta (γδ) T lymphocytes in the peripheral blood of crossbreed dogs during stages of life and implication in aging.

BACKGROUND: Data on gamma-delta (γδ) T lymphocytes in the peripheral blood of dogs are scant, related only to healthy pure breed dogs and limited to a restricted age range. The aim of the study was to investigate the modulation of the γδ T lymphocyte (TCRγδ+) subpopulation in peripheral blood of crossbreed healthy dogs according to five identified stages of life: Puppy, Junior, Adult, Mature, Senior and to determine its implication in aging. A rigorous method of recruitment was used to minimize the influence of internal or external pressure on the immune response. Twenty-three intact female and twenty-four intact male dogs were enrolled. Blood samples were collected and immunophenotyping of peripheral blood T lymphocytes and γδ T cell subpopulations was performed. RESULTS: The percentage of γδ T cells in peripheral blood lymphocytes was comparable with the value of 2.5% published by Faldyna and co-workers (2001), despite the percentage reported was investigated in less arranged age range groups and coming from four different dog pure breeds, whereas our data were recorded on wider age range groups and coming from crossbreed dogs. Therefore, the γδ T cell percentage (2.5%) is consistent and points out that such value is breed-independent. Statistical analysis highlighted differences in both percentage and absolute γδ T cells according to the stage of life. γδ T cells decreased significantly in the peripheral blood of elder dogs (Senior group) in comparison with previous stages of life (Puppy, Junior, and Adult groups). Differences in γδ T cells are significant and they are reported, for the first time, related to dog aging. CONCLUSIONS: The study confirms dogs to be among the animals with a low TCRγδ+ cell profile. A decrease of the TCRγδ+ subpopulation percentage was observed in elder dogs. TCRγδ+ cells of group S were different from those of groups P, J, and A. The differences are reported for the first time in dog aging. Identifying the stage of life when the decrease of γδ T lymphocytes starts can be useful for providing a rationale for drafting a wellness plan trial to support thymus immune functions and mitigate its functional exhaustion.

In vitro and in vivo characterization of poractant alfa supplemented with budesonide for safe and effective intratracheal administration.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

The integration of orthodontic miniscrews under mechanical loading: a pre-clinical study in rabbit.

INTRODUCTION: Orthodontic miniscrews are an increasingly popular choice to achieve absolute anchorage. The temporary use of miniscrews and their recent introduction have limited the debate over the biological aspect of the materials to that of the surface that permeates the field of dental implants. The aim of the present study was to investigate the integration of grade 5 titanium mini-implants with machined or sand blasted acid etched surface (SAE) under mechanical load in a rabbit tibia model of implant integration. METHODS: A total of 64 miniscrews (Ti6Al4V) of 1.5 mm diameter and 6.5 mm length were inserted in the proximal medial surface of each tibia in eight male rabbits aged 6 months. Each tibia received four miniscrews. A 100 g nickel-titanium coil spring (Neosentalloy) was applied between two miniscrews along the main axis while two miniscrews were left unloaded. The removal torque was measured for loaded and unloaded miniscrews after 12 weeks. Two miniscrews were harvested for histology. RESULTS: Removal torque was significantly higher for SAE mini-implants than for machined screws, under both loading conditions. Although no difference in bone to implant contact was observed among the groups, cortical area significantly decreased with both surfaces under loading. CONCLUSIONS: Our data indicate that SAE miniscrews have higher bone retention than MA miniscrews, although the effects of mechanical loading of these devices on cortical bone require further investigations.

Morphological analysis of the urethral muscle of the male pig with relevance to urinary continence and micturition.

To investigate whether the pig could be considered a suitable model to study lower urinary tract function and dysfunction, the pelvic urethra of 24 slaughtered male pigs were collected, and the associated muscles were macroscopically, histologically and histochemically analyzed. In cross-sections of the urethra, a muscular complex composed of an inner layer of smooth muscle and an outer layer of striated muscle that are not separated by fascial planes was observed. A tunica muscularis, composed of differently oriented smooth muscle bundles, is only evident in the proximal part of the pelvic urethra while, in the remaining part, it contributes to form the prostatic fibromuscular stroma. The striated urethral muscle surrounds the pelvic urethra in a horseshoe-like configuration with a dorsal longitudinal raphe, extending from the bladder neck to the central tendon of perineum. Proximally to the bladder, it is constituted of slow-twitch and fast-twitch myofibers of very small diameter, and embedded in an abundant collagen and elastic fiber net. Moving caudally it is gradually encircled and then completely substituted by larger and compact myofibers, principally presenting circular orientation and fast-twitch histochemical characteristics. So, like in humans, the cranial tract of the muscular system surrounding the pelvic urethra is principally composed of smooth musculature. The striated component cranially may have a role in blocking retrograde ejaculation, while the middle and caudal tracts may facilitate urine and semen flow, and seem especially concerned with the rapid and forceful urethral closure during active continence. Some differences in the morphology and structure between pigs and humans seem due to the different morphology of the 'secondary' sexual organs that develop from the urethral wall and to the different effect of gravity on the mechanics of the urinary system in quadruped and bipedal mammals.

Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: an in vivo study.

BACKGROUND: Osteochondral defects significantly affect patients' quality of life and represent challenging tissue lesions, because of the poor regenerative capacity of cartilage. Tissue engineering has long sought to promote cartilage repair, by employing artificial scaffolds to enhance cell capacity to deposit new cartilage. An ideal biomaterial should closely mimic the natural environment of the tissue, to promote scaffold colonization, cell differentiation and the maintenance of a differentiated cellular phenotype. The present study evaluated chitosan scaffolds enriched with D-(+) raffinose in osteochondral defects in rabbits. Cartilage defects were created in distal femurs, both on the condyle and on the trochlea, and were left untreated or received a chitosan scaffold. The animals were sacrificed after 2 or 4 weeks, and samples were analysed microscopically. RESULTS: The retrieved implants were surrounded by a fibrous capsule and contained a noticeable inflammatory infiltrate. No hyaline cartilage was formed in the defects. Although defect closure reached approximately 100% in the control group after 4 weeks, defects did not completely heal when filled with chitosan. In these samples, the lesion contained granulation tissue at 2 weeks, which was then replaced by fibrous connective tissue by week 4. Noteworthy, chitosan never appeared to be integrated in the surrounding cartilage. CONCLUSIONS: In conclusion, the present study highlights the limits of D-(+) raffinose-enriched chitosan for cartilage regeneration and offers useful information for further development of this material for tissue repair.

Effect of different atmospheric and subatmospheric cooking techniques on physical and chemical qualitative properties of pork loin.

In this paper, the effects of four cooking procedures were evaluated, two occurring in atmospheric (in ventilated and steam oven) and two in subatmospheric (vacuum and sous vide cooking) conditions on pork Longissimus lumborum. The main objective of the study was to compare and evaluate the physical and chemical characteristics. Samples were cooked in four independent trials namely Oven (O), Steaming (ST), Vacuum Cooking (VC) and Sous Vide (SV). The analyses included temperature, cooking effect, percentage weight loss, texture (cutting and double compression tests), colour (superficially and inside the sample), microstructure (optical microscopy) and fibres shortening analysis. To assess cooking effects on significant nutritional constituents, the fatty acid composition and the content of B vitamins were analysed. Volatile profiles of samples were also compared using solid-phase microextraction. SV cooking resulted in the less favourable meat texture, presenting the highest hardness and chewiness. Moreover, high hardness values measured on SV samples is also related to the high weight loss. The technique of oven cooking (O) demonstrated superior results in terms of mechanical properties, which are closely associated with the cooking values. Specifically, the cook value C0 was significantly higher in the case of oven cooking compared to SV, VC, and ST. Mild temperature conditions and cooking times of the four considered cooking techniques did not induce significant variations in the fatty acid composition and volatile profile. Conversely, SV and VC allowed the highest amount of vitamin B retention in cooked meat. This work suggests that some differences emerged on the effects due to sub-atmospheric and atmospheric cooking compared to traditional ones.

Preterm rabbit-derived Precision Cut Lung Slices as alternative model of bronchopulmonary dysplasia in preclinical study: a morphological fine-tuning approach.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm delivery, with significant morbidity and mortality in a neonatal intensive care setting. Research in this field aims to identify the mechanisms of late lung development with possible therapeutic targets and the improvement of medical management. Rabbits represent a suitable lab preclinical tool for mimicking the clinical BPD phenotype. Rabbits are born at term in the alveolar phase as occurs in large animals and humans and in addition, they can be delivered prematurely in contrast to mice and rats. Continuous exposure to high oxygen concentration (95% O2) for 7 days induces functional and morphological lung changes in preterm rabbits that resemble those observed in BPD-affected babies. The preclinical research pays great attention to optimize the experimental procedures, reduce the number of animals used in experiments and, where possible, replace animal models with alternative assays, following the principle of the 3 Rs (Replace, Reduce and Refine). The use of in vitro assays based on the ex vivo culture of Precision Cut Lung Slices (PCLS) goes in this direction, representing a good compromise between controlled and flexible in vitro models and the more physiologically relevant in vivo ones. This work aims to set up morphological analyses to be applied in preclinical tests using preterm rabbits derived PCLS, cultured up to 7 days in different oxygen conditions, as a model. After a preliminary optimization of both lung preparation and histological processing methods of the lung slices of 300 µm, the morphological analysis was conducted evaluating a series of histomorphometric parameters derived from those widely used to follow the phases of lung development and its alterations in vivo. Our histomorphometric results demonstrated that the greatest differences from pseudo-normoxia and hyperoxia exposed samples at day 0, used as starting points to compare changes due to treatments and time, are detectable after 4 days of in vitro culture, representing the most suitable time point for analysis in preclinical screening. The combination of parameters suitable for evaluating PCLS morphology in vitro resulted to be Tissue Density and Septal Thickness. Shape Factor and Roughness, evaluated to highlight the increasing complexity of the airspaces, due to the formation of septal crests, gave useful information, however, without significant differences up to day 4. Other parameters like Mean Linear Intercept and Septal Density did not allow to highlight significant differences between different oxygen conditions and time points. Instead, Radial Alveolar Count, could not be applied to PCLS, due to the tissue changes following agar infusion and culture conditions.

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.

Biofunctionalization of bioactive ceramic scaffolds to increase the cell response for bone regeneration.

Biofunctionalization was investigated for polymers and metals considering their scarce integration ability. On the contrary few studies dealt with ceramic biofunctionalization because the bioactive and bioresorbable surfaces of ceramics are able to positively interact with biological environment. In this study the cell-response improvement on biofunctionalized wollastonite and diopside-based scaffolds was demonstrated. The ceramics were first obtained by heat treatment of a silicone embedding reactive oxide fillers and then biofunctionalized with adhesive peptides mapped on vitronectin. The most promisingin vitroresults, in terms of h-osteoblast proliferation and bone-related gene expression, were reached anchoring selectively a peptide stable toward proteolytic degradation induced by serum-enriched medium. Inin vivoassays the anchoring of this protease-stable adhesive peptide was combined with self-assembling peptides, for increasing cell viability and angiogenesis. The results demonstrated external and internal cell colonization of biofunctionalized scaffolds with formation of new blood vessels (neoangiogenesis) and stimulation of ectopic mineralization.

Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces.

OBJECTIVE: The aim of this study is to analyze the morphology and proliferation of human osteoblastic cells in vitro on five commercially available titanium surfaces. MATERIALS AND METHODS: Human primary cells of the osteoblastic lineage were obtained from bone explants. The cells were plated on polished (T1), machined (T2), sand-blasted/acid-etched (T3), sand-blasted/acid-etched, modified with hydrogen peroxide rinse (T4), and plasma-sprayed titanium (T5) disks. Cell morphology was studied after 6, 24, 72 h, 7 and 14 days of culture by scanning electron microscopy. The formation and distribution of focal adhesions was investigated by immunocytochemical staining at 3, 6 and 24 h. Cell growth was measured by an MTT assay after 3, 7 and 9 days of culture. Moreover, the production of osteocalcin and osteoprotegerin (OPG) was evaluated in the supernatants by ELISA. RESULTS: Morphological analysis revealed that substrate topography profoundly affected cells' shape and their anchoring structures. Large lamellipodia were formed on polished and machined surfaces, while thin filopodia were more frequently observed on T3 and T4 samples. Moreover, cells formed stronger focal adhesions on T3 and T4 surfaces, and cell proliferation was higher on rough surfaces. Osteocalcin production was higher on the T4 surface, whereas OPG steadily increased on every surface. CONCLUSIONS: Taken together, these data show that all the surfaces allowed cell attachment, adhesion and proliferation, but T4 and T5 surfaces appeared to be a better substrate for the adhesion, proliferation and differentiation of cells of the osteoblastic lineage.

Gene expression markers in horse articular chondrocytes: Chondrogenic differentiaton IN VITRO depends on the proliferative potential and ageing. Implication for tissue engineering of cartilage.

Chondrocyte dedifferentiation is a key limitation in therapies based on autologous chondrocyte implantation for cartilage repair. Articular chondrocytes, obtained from (metacarpophalangeal and metatarsophalangeal) joints of different aged horses, were cultured in monolayer for several passages (P0 to P8). Cumulative Populations Doublings Levels (PDL) and gene expression of relevant chondrocyte phenotypic markers were analysed during culturing. Overall data confirmed that, during proliferation in vitro, horse chondrocytes undergo marked morphological and phenotypic alterations of their differentiation status. Particularly, the dedifferentiation started early in culture (P0-P1) and was very marked at P3 subculture (PDL 4-6): proliferative phase after P3 could be critical for maintenance/loss of differentiation potential. In elderly animals, chondrocytes showed aspects of dedifferentiation shortly after their isolation, associated with reduced proliferative capacity. Regarding the gene expression of major cartilage markers (Col2, Aggrecan, SOX9) there was a very early reduction (P1) in proliferating chondrocytes independent of age. The chondrocytes from adult donors showed a more stable expression (up to P3) of some (Col6, Fibromodulin, SOX6, TGß1) markers of mature cartilage; these markers could be tested as parameter to determine the dedifferentiation level. This study can provide parameters to identify up to which "culture step" chondrocytes for implantation with a conserved phenotypic potential can be obtained, and to test the efficiency of biomaterial scaffold or chondroinductive media/signals to maintain/recover the chondrocyte phenotype. Moreover, the determination of levels and time related expression of these markers can be useful during the chondroinduction of mesenchymal stem cells.

Inflammatory Cytokines and Biodegradable Scaffolds in Dental Mesenchymal Stem Cells Priming.

Mesenchymal stem cells (MSCs) are multipotent stem cells with wide-ranging clinical applications due to their ability to regenerate tissue from mesenchymal origin and their capability of suppressing immune responses, thus reducing the likelihood of graft versus host disease after transplantation. MSCs can be isolated from a variety of sources including bone marrow, adipose tissue, umbilical cord blood, and immature teeth. Dental stem cells (DSCs) possess progenitor and immunomodulatory abilities as the other MSC types and because they can be easily isolated, are considered as attractive therapeutic agents in regenerative dentistry. Recently, it has been shown that DSCs seeded onto newly developed synthetic biomaterial scaffolds have retained their potential for proliferation and at the same time have enhanced capabilities for differentiation and immunosuppression. The scaffolds are becoming more efficient at MSC priming as researchers learn how short peptide sequences alter the adhesive and proliferative capabilities of the scaffolds by stimulating or inhibiting classical osteogenic pathways. New findings on how to modulate the inflammatory microenvironment, which can prime DSCs for differentiation, combined with the use of next generation scaffolds may significantly improve their therapeutic potential. In this review, we summarize current findings regarding DSCs as a potential regenerative therapy, including stem cell priming with inflammatory cytokines, types of scaffolds currently being explored and the modulation of scaffolds to regulate immune response and promote growth.

Striated Perineal Muscles: Location of Somatic and Autonomic Neurons Projecting to the Male Pig Ischiocavernous Muscle. Neurochemical Features of the Sympathetic Subset.

The location, number and size of the central and peripheral neurons innervating the ischiocavernous muscle (ICM) were studied in male pigs by means of Fast Blue (FB) retrograde neuronal tracing. Moreover the immunohistochemical properties of the sympathetic ganglia were investigated combining the double immunolabeling method. After injection of FB into the left ICM, a mean number of 245.3 ± 134.9 labeled neurons were found in the ipsilateral ventral horn of the S1-S3 segments of the spinal cord (SC), 129.7 ± 45.5 in the L6-S3 ipsilateral and S2-S3 contralateral spinal ganglia (SGs), 2279.3 ± 622.1 in the ipsilateral L2-S2 and contralateral L5-S2 sympathetic trunk ganglia (STGs), 541.7 ± 158 in the bilateral caudal mesenteric ganglia (CMGs), and 78.3 ± 35.8 in the microganglia of the pelvic plexus (PGs). The mean area of the ICM projecting neurons was 1217 ± 69.7 µm2 in the SC, 2737.5 ± 176.5 µm2 in the SGs, 982.8 ± 36.8 µm2 in the STGs, 865.9 ± 39.14 µm2 in the CMGs and 426.2 ± 24.72 µm2 in the PGs. The FB positive neurons of autonomic ganglia contained Dopamine ß hydroxylase, vesicular acetylcholine transporter, neuronal nitric oxyde sinthase, calcitonine gene related peptide, leu-enkephaline, neuropeptide Y, substance P, vasoactive intestinal polypeptide, and somatostatine often colocalized with tyrosine hydroxylase. The particular localization of the motor somatic nucleus, the abundant autonomic innervation and the qualitatively different content of ICM projecting sympathetic neurons suggest a complex regulation of this striated muscle involved in involuntary functions, such as the erection, ejaculation, micturition and defecation. Anat Rec, 301:837-848, 2018. © 2017 Wiley Periodicals, Inc.

Immunolocalization of Orexin A and its receptors in the different structures of the porcine ovary.

Orexins are neuropeptides with pleiotropic functions, involved in the coordination of multiple versatile physiological processes, in particular related to food intake and several aspects of the reproductive process. Their actions are carried out through the bond with the related Orexin 1 (OXR1) and Orexin 2 (OXR2) G-protein-coupled receptors. Studies on the expression of the orexinergic system in the female genital organs are scarce and limited to preovulatory gametogenic follicles and corpora lutea isolated from the rest of the ovary. As the description of only these structures is insufficient to provide a complete picture of the organ, the present study is aimed to give a panoramic view of all the ovarian structures and cells expressing Orexin A (OXA) and its receptors in their original localization. Double labeling immunofluorescent methods, applied on frozen sections of the whole organ in both follicular and luteal phase, were used to highlight the particular distribution and colocalization of the proteins. For a better recognition of cellular morphology and a better distinction between gametogenic (healthy) and atretic follicles, also a single labeling immunolocalization of OXA on formalin fixed paraffin embedded tissues and a TUNEL staining were performed. The results indicate that OXA and its two receptors subtypes are expressed in all the different structures composing the swine ovary, albeit in different ways, in both phases of the ovarian cycle. In general, OXA and OXR2 appear diffusely distributed within "health", proliferating and steroid producing cells, while has granular appearance, being presumably associated to cytoplasmic vesicles, in degenerating cells, independently if apoptotic or not. The immunoreactivity for OXR1, instead, is often associated with the nuclear envelope but it is also detectable, to a lesser extent, diffusely distributed in the cytoplasm of growing or steroid producing cells. When cells undertake the path leading to degeneration, also OXR1 immunoreactivity assumes a granular appearance in the cytoplasm and is colocalized with OXA and OXR2. Different roles for the two receptors in the same cell and a different regulation of their expression remain to be investigated. Their comprehension could help studies of follicle development in pig, as part of in vitro oocyte maturation and fertilization programs in livestock.

Surfactant replacement therapy in combination with different non-invasive ventilation techniques in spontaneously-breathing, surfactant-depleted adult rabbits.

Nasal intermittent positive pressure ventilation (NIPPV) holds great potential as a primary ventilation support method for Respiratory Distress Syndrome (RDS). The use of NIPPV may also be of great value combined with minimally invasive surfactant delivery. Our aim was to implement an in vivo model of RDS, which can be managed with different non-invasive ventilation (NIV) strategies, including non-synchronized NIPPV, synchronized NIPPV (SNIPPV), and nasal continuous positive airway pressure (NCPAP). Forty-two surfactant-depleted adult rabbits were allocated in six different groups: three groups of animals were treated with only NIV for three hours (NIPPV, SNIPPV, and NCPAP groups), while three other groups were treated with surfactant (SF) followed by NIV (NIPPV+SF, SNIPPV+SF, and NCPAP+SF groups). Arterial gas exchange, ventilation indices, and dynamic compliance were assessed. Post-mortem the lungs were sampled for histological evaluation. Surfactant depletion was successfully achieved by repeated broncho-alveolar lavages (BALs). After BALs, all animals developed a moderate respiratory distress, which could not be reverted by merely applying NIV. Conversely, surfactant administration followed by NIV induced a rapid improvement of arterial oxygenation in all surfactant-treated groups. Breath synchronization was associated with a significantly better response in terms of gas exchange and dynamic compliance compared to non-synchronized NIPPV, showing also the lowest injury scores after histological assessment. The proposed in vivo model of surfactant deficiency was successfully managed with NCPAP, NIPPV, or SNIPPV; this model resembles a moderate respiratory distress and it is suitable for the preclinical testing of less invasive surfactant administration techniques.

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure. The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets.

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase-independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathione levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS-, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL-2 production. Overall, our data demonstrate that HS- reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL-2, therefore de-activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti-inflammatory effects of sulfur compounds.

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies.

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29+, CD44+, CD73+, CD90+, CD34-, CD45- and MHC-II- with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90+, CD73+, CD105+, CD44+, CD13+, CD29+, Oct-4+ gene and CD31- and CD45- expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures.