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The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

The occurrence of the honeybee caste polyphenism arises when a change in diet is transduced into cellular metabolic responses, resulting in a developmental shift mediated by gene expression. The aim of this investigation was to detect and describe the expression profile of water-soluble proteases during the ontogenesis of honeybee worker-fate larvae. The extraction of insect homogenates was followed by the electrophoretic separation of the protein extract in polyacrylamide gels under semi-denaturing condition, precast with gelatin, pollen, or royal jelly protein extracts. The worker-fate honeybee larva showed a proteolytic pattern that varied with aging, and a protease with the highest activity at 72 h after hatching was named PS4. PS4 has a molecular weight of 45 kDa, it remained active until cell sealing, and its enzymatic properties suggest a serine-proteinase nature. To define the process that originates a queen-fate larvae, royal jelly and pollen were analysed, but PS4 was not detected in either of them. The effect of food on the PS4 was investigated by mixing crude extracts of queen and worker-fate larvae with pollen and royal jelly, respectively. Only royal jelly inhibited PS4 in worker-fate larvae. Taken together, our data suggest that PS4 could be involved in caste differentiation.

Fishmeal (FM) is still the most important protein source in aquafeeds. However, due to the reduction of wild fish stocks used for FM production, its manufacturing it is now unsustainable. Insect meal represents a valid alternative to FM, due to the low carbon footprint of its production and its high nutritional value. The aim of this study was to investigate the potentials of replacing FM with black soldier fly (Hermetia illucens) meal (HIM) in aquafeeds, using zebrafish as the animal model. Four diets were formulated with increasing HIM/FM replacement rate (the control diet contained 20% FM). Mortality rates, growth performances, and feed consumptions were measured, and fish intestine samples were collected for histological and enzymatic analysis. After 49 days, all groups almost tripled their initial body weight (cumulative body weight gain ranged between 143.9 and 155.2 mg), and no statistically significant differences among treatments were observed in relation to growth performances and histological traits. Contrarily, trypsin, alkaline phosphatases, and alpha amylase–glucoamylase activities were significantly reduced when the FM replacement rate was increased. In conclusion, HIM may represent an alternative to FM, since no adverse effects were observed when it was included up to 20% in the zebrafish diet, even when replacing 100% of the FM.

The aim of the present study was to compare the effects of two different nitrogen-rich ingredients such as hydrolysed fish protein and autolysed yeast, on gilthead sea bream (Sparus aurata) growth performances and histological gut morphology. Animals were allocated to three experimental groups: the first received a fishmeal-based diet (FM), the second and the third received hydrolysed fish protein (HFP) and autolysed yeast (AY), respectively, to replace an equal amount of fishmeal. The experiment lasted 92 days. No significant differences in body weight and mortality were observed among the different groups. Villus branching, intraepithelial lymphocytes and inflammatory infiltrate of the submucosa were more prominent in AY than HFP and FM. The gut absorbent surface ratio was 5.94, 6.44 and 7.28 for group FM, HFP and AY, respectively, with statistical significant difference between FM and AY and between HFP and AY. A significant increment in the goblet cell density was observed for HFP and AY in comparison to FM. A statistically significant increase in small-sized goblet cells was observed in AY compared to FM. All our findings suggest that it is possible to use either HFP or AY, as partial replacer of FM in the S. aurata diet.Highlights Yeast-derived products as a suitable alternative nitrogen source. Autolyzed yeast as a novel approach in the use of yeast products. Use of autolysed yeast for replacing fishmeal in aqua feed. Use of hydrolysed fish protein for replacing fishmeal in aqua feed.

The domestic pig (Sus scrofa domesticus) stems from the Eurasian wild boar (Sus scrofa): this offers an appealing window to study microanatomical changes related to the process of domestication, the symbiotic relationship between human and animal. In this light, we quantitatively demonstrated significant microanatomical differences between pig and wild boar cerebella. Calbindin D-28, a calcium binding protein, was employed as immunohistochemical marker of the Purkinje cells. Our results showed that: (i) the foliation index, expressing the rate of cerebellar cortical folding, and the number of granular cells were not significantly different between pigs and wild boars; (ii) area of the granular layer and the molecular layer, and area of white matter were lower in pigs; (iii) the fraction area, grey matter/white matter, was higher in pigs; (iv) the Purkinje cell linear density and their soma area were higher in wild boars. Despite the morphological data alone are not sufficient to draw any final conclusions, our findings on Purkinje cells may represent good indicators of a reduction of the pig cerebellum motor and cognitive functions during the process wild boar-to-pig domestication.

The canine placenta is an underexamined organ. Placental abnormalities can affect foetus development and may be responsible for a low weight of the infant at birth; however, knowledge on their clinical significance in the canine species is limited. We aimed to describe macroscopic and microscopic findings in the canine placenta and amnion at term in clinically uncomplicated pregnancies and to evaluate their relationship with birth weight of healthy puppies. During natural delivery or C‐section, the birth weight of 82 puppies was recorded, 72 placentas and 66 amnions were recovered. The foetal and maternal surfaces of the placental girdle, marginal haematoma and amnion were evaluated. Each gross finding was recorded, morphometrically assessed and sampled for histological diagnosis. Furthermore, specimens of placenta and amnion were collected from representative areas and microscopic deviations from normal structure were evaluated in haematoxylin and eosin sections. Gross examination revealed ‘abnormalities’ in the 75.4% of the collected placentas. Necrosis was the gross change most commonly observed in the placental girdle (72.5%). Congestion (17.4%) and clotted blood/fibrinoid material (2.9%) were also observed. No gross changes of either the marginal haematoma or the amnion were recorded. Histologically, placental girdle showed necrosis (62.3%), mineralization (52.2%), congestion (36.2%) and neutrophilic infiltration (27.5%). Marginal haematoma exhibited mineralization (11.6%) and neutrophils (29%), while necrotic foci were rarely observed (4.3%). In the amnion, the most frequent alteration observed was hypertrophy of the epithelium (35.9%) followed by oedema (31.2%), mineralized foci (28.1%), fibrosis (23.4%), congestion (15.6%) and more rarely neutrophils (12.5%). Puppies’ birth weight was not statistically affected by either gross or histological abnormalities. Our study revealed that macroscopic and microscopic ‘abnormalities’ of the placenta and amnion may be common in uncomplicated pregnancies at term; however, no implications on puppies’ birth weight were observed. Deviations from ‘normal’ morphology of canine foetal adnexa warrant further investigation to assess their clinical implications if present.

Trigeminal input exerts acute and chronic effects on the brain, modulating cognitive functions. Here, new data from humans and animals suggest that these effects are caused by trigeminal influences on the Locus Coeruleus (LC). In humans subjects clenching with masseter asymmetric activity, occlusal correction improved cognition, alongside with reductions in pupil size and anisocoria, proxies of LC activity and asymmetry, respectively. Notably, reductions in pupil size at rest on the hypertonic side predicted cognitive improvements. In adult rats, a distal unilateral section of the trigeminal mandibular branch reduced, on the contralateral side, the expression of c-Fos (brainstem) and BDNF (brainstem, hippocampus, frontal cortex). This counterintuitive finding can be explained by the following model: teeth contact perception loss on the lesioned side results in an increased occlusal effort, which enhances afferent inputs from muscle spindles and posterior periodontal receptors, spared by the distal lesion. Such effort leads to a reduced engagement of the intact side, with a corresponding reduction in the afferent inputs to the LC and in c-Fos and BDNF gene expression. In conclusion, acute effects of malocclusion on performance seem mediated by the LC, which could also contribute to the chronic trophic dysfunction induced by loss of trigeminal input.

The endocannabinoid system—or the broader endocannabinoidome—is an emerging key player in skin homeostasis to the extent that it has recently been termed “c(ut)annabinoid system”. Receptors, mediators, and regulatory molecules are produced/expressed by most skin cellular elements and a plethora of intricate mechanisms are increasingly being acknowledged to explain the endocannabinoidome role in skin homeostasis. Among skin cells, mast cell function is regulated by N-acylethanolamines, among which palmitoylethanolamide (PEA) is the most studied molecule. The purpose of this chapter is to review the body of evidence on the endocannabinoidome’s role in skin physiology and pathology, with particular reference to canine and feline species. The detection of mediators and receptors in the skin will be reviewed. The main dermatological preclinical and clinical data from studies involving animals treated with endocannabinoids and related N-acylethanolamines will also be presented since there is a paucity of clinical evidence utilizing exogenous cannabinoids thus far. Skin allergy represents a promising target for endocannabinoid treatment in the field of veterinary dermatology.

Contamination by organic and inorganic compounds remains one of the most complex problems in both brackish and marine environments, causing potential implications for the reproductive success and survival of several broadcast spawners. Ficopomatus enigmaticus is a tubeworm polychaete that has previously been used as a model organism for ecotoxicological analysis, due to its sensitivity and ecological relevance. In the present study, the effects of five trace elements (zinc, copper, cadmium, arsenic and lead), one surfactant (sodium dodecyl sulfate, SDS) and one polycyclic aromatic hydrocarbon (benzo(a)pyrene, B(a)P) on the sperm quality of F. enigmaticus were investigated. Sperm suspensions were exposed in vitro to different concentrations of each selected contaminant under four salinity conditions (10, 20, 30, 35). Possible adverse effects on sperm function were assessed by measuring oxidative stress, membrane integrity, viability and DNA damage. Sperm quality impairments induced by organic contaminants were more evident than those induced by inorganic compounds. SDS exerted the largest effect on sperm. In addition, F. enigmaticus sperm showed high tolerance to salinity variation, supporting the wide use of this species as a promising model organism for ecotoxicological assays. Easy and rapid methods on polychaete spermatozoids were shown to be effective as integrated sperm quality parameters or as an alternative analysis for early assessment of marine and brackish water pollution.

Puppy neonatal mortality may be related to low birth weight, which has been shown in humans to be linked to placental factors. The relation between the newborn puppy and the placental characteristics has been poorly investigated in dogs. Twenty bitches, 9 toy-sized (i.e.

The claustrum (Cl) is a subcortical nucleus present in all mammalian species examined so far, whose function is still a puzzling problem. There is a wealth of data on its general anatomy, cytoarchitecture, and chemoarchitecture including the connectivity with both cortical and subcortical structures; instead, much less is known about the presence of the endocannabinoid system (ECs) an important neuromodulatory complex in this brain area. In an attempt to better understand the role of the ECs within the Cl circuitry, we undertook an immunohistochemical analysis to describe the distribution of the CB1 and of the endogenous cannabinoids degrading enzymes MGL and FAAH in the pig Cl as well as their relationship with both the catecholaminergic system and with parvalbumin (PV) expressing neurons. CB1, FAAH and MGL were expressed throughout the entire Cl. CB1 was expressed by fibers and puncta, while FAAH and MGL were mainly localized in the neuropil. FAAH also showed a faint cell body localization that colocalized with PV. Tyrosine hydroxylase positive fibers (catecholaminergic system), did not demonstrate the presence of CB1. Taken together, the results described herein provide evidence for an anatomical distribution of a CB1/PV signaling system in the pig Cl suggesting that PV cells may play a role within the ECs.

Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. In particular, it is implicated in ageing-related diseases, such as cardiac hypertrophy, myocardial infarct, and endothelial dysfunction; moreover, its expression decreases with age. Therefore, an effective strategy to extend the lifespan and improve cardiovascular function is the enhancement of the expression/activity of SIRT1 with exogenous agents. The Citrus flavonoid naringenin (NAR) presents structural similarity with the natural SIRT1 activator resveratrol. In this study, we demonstrate through in vitro assays that NAR significantly activates SIRT1 enzyme and shows antisenescence effects. The binding mode of NAR into SIRT1 was detailed investigated through in silico studies. Moreover, chronic administration (for six months) of NAR (100 mg/kg/day) to 6-month-old mice leads to an enhancement of SIRT1 expression and a marked reduction of reactive oxygen species production in myocardial tissue. Furthermore, at the end of the treatment, the plasma levels of two well-known markers of cardiovascular inflammation, TNF-α and IL6, are significantly reduced in 12-month-old mice treated with NAR, as well as the cardiovascular risk (total cholesterol/HDL ratio) compared to control mice. Finally, the age-associated fibrotic remodeling, which is well detected through a Mallory trichrome staining in the vehicle-treated 12-month-old mice, is significantly reduced by the chronic treatment with NAR. Moreover, an improvement of myocardium functionality is highlighted by the enhancement of citrate synthase activity and stabilization of the mitochondrial membrane potential after NAR treatment. Taken together, these results suggest that a nutraceutical approach with NAR may have positive impacts on many critical hallmarks of myocardial senescence, contributing to improve the cardiac performance in aged subjects.

Obesity is currently considered a major source of morbidity, with dramatic complications on health status and life expectancy. Several studies demonstrated the positive effects of Brassicaceae vegetables on obesity and related diseases, partially attributing these beneficial properties to glucosinolates and their derivatives isothiocyanates. Recently, isothiocyanates have been described as a hydrogen sulfide (H2 S)-releasing moiety, suggesting that H2 S may be at least in part responsible for the beneficial effects of Brassicaceae. In this work, the metabolic effects of an extract obtained from Eruca sativa Mill. seeds (E.S., Brassicaceae), containing high levels of glucoerucin, were evaluated in an experimental model of obesity. Male balb/c mice were fed for 10 weeks with standard (Std) diet or high fat (HF) diet supplemented with E.S. E.S. significantly contained the body weight gain in this obesity model, improving also glucose homeostasis. Interestingly, lower values of white adipose tissue mass and a significant reduction of adipocytes size were also observed. Moreover, E.S. enhanced the adipocytes metabolism, improving the citrate synthase activity and reduced triglyceride levels in mice fed with HF diet. Taken together, these results suggest that E.S. is endowed with an interesting translational and nutraceutical value in the prevention of metabolic disorders, suggesting that H2 S could be a key player.

This study aimed at selecting effective histological indicators of the freezing process, for the discrimination of fresh and frozen common octopus Octopus vulgaris. Histological indices of freezing process were selected in mantle and arm muscle and axial nerve tissues. Seven histological parameters were chosen: overall muscle tissue structural organization (a); gaping among muscle bundles (b); presence of optically empty spaces between and within muscle bundles (c); white spaces percentage between and within muscle bundles (d); overall nerve structural organization (e); presence of linear fissures and/or empty spaces within neuropil and axonal tract (f); presence of empty spaces within connective tissue matrix surrounding the nerve (g); empty space percentage within arm axial nerve region (h). The parameters were assessed on 150 mantle muscle sections (a, b, c, d), 150 arm muscle (b, c) and nervous (e, f, g, h) tissue sections belonging to 20 fresh exemplars further subdued to conventional freezing procedure at − 20 °C, 25 fresh curled exemplars, 25 exemplars industrially frozen at − 80 °C, and 20 thawed, curled, and individually quick frozen exemplars. Overall structural organization (a, e), gaping (b), and presence of optically empty spaces (c, f) confirmed significant morphological freezing indices in mantle and arm sections. In mantle, two d values (d

Cannabinoids help in pain treatment through their action on CB1 and CB2 receptors. β-caryophyllene (BCP), an ancient remedy to treat pain, is a sesquiterpene found in large amounts in the essential oils of various spice and food plants such as oregano, cinnamon, and black pepper. It binds to the CB2 receptor, acting as a full agonist. Sex differences in the BCP-induced analgesic effect were studied by exposing male and female rats to a persistent/repeated painful stimulation. To simulate treatment of a repeated inflammatory condition, after the first formalin injection (FT1; 50 μl, 2.5%), rats received BCP per os for 7 days at two dosages: 5 and 10 mg/kg dissolved in olive oil (OIL). The control group was treated with OIL for 7 days. On day 8, the formalin test was repeated (FT2) with a lower formalin concentration (50 μl, 1%). During the first and second formalin tests, pain-induced responses (licking, flexing, and paw jerk) and spontaneous behaviors were recorded and analyzed. In the FT1 (before the beginning of treatment with BCP), females displayed higher pain responses than did males in terms of flexing duration during the first part of the test (I phase and interphase), while during the second part (II phase early and late) males showed higher levels than did females in licking duration. In the FT2, the pain responses generally decreased in the BCP groups in a dose-dependent manner (i.e., greater effect of BCP10), with a more pronounced reduction in males than in females; moreover, the pain responses remained high in the OIL groups and in the female BCP5 group. In conclusion, long-term intake of BCP appears to be able to decrease pain behaviors in a model of repeated inflammatory pain in both sexes, but to a greater degree in males.

Information on microscopic anatomy of equine skin is sparse. In horses, limb wounds often become chronic and/or non-healing whereas body wounds heal normally. These dissimilarities in healing patterns might be a product of different phenotypic characteristics of body and limb skin. The objective of this study was to investigate microscopic anatomy, epidermal thickness, keratinocyte proliferation and differentiation as well as the presence of mast cells in normal equine skin of body and limb.The study involved body and limb skin biopsies from six horses. Histological characteristics of the epidermis were assessed and epithelial thickness measured. Immunohistochemistry was performed to investigate epidermal differentiation patterns of cytokeratin (CK) 10, CK14, CK16, loricrin, and peroxisome proliferator-activated receptor alpha (PPAR-α), epidermal proliferation (Ki-67 immunostaining), and mast cells distribution in the skin.The epidermis was significantly thicker in the limb skin compared to body skin (p0.01). Epidermal proliferation and CK distribution did not show differences in the two anatomical areas. Loricrin presence was focally found in the spinous layer in four out of six limb skin samples but not in body skin samples. Tryptase positive mast cells were detected in the dermis and their density (cell/mmHere we report for the first time about the normal distribution of CK10, CK14, CK16, PPAR-α, and loricrin in equine limb and body skin as well as about epidermal proliferation rate and mast cell count. It will be relevant to investigate the distribution of the investigated epithelial differentiation markers and the role of mast cells during equine wound healing and/or other skin diseases.

Simple Summary DDX3X has been repeatedly proven to exert pro-tumorigenic effects in breast cancer. An inhibitor of DDX3X helicase function, FHP01, exhibited very effective antiproliferative and tumor killing activity in vitro and in vivo against several breast cancer cells, yet showed no systemic toxicity but good bioavailability in vivo. FHP01 may therefore represent a novel therapeutic approach with high potential as a personalized strategy for breast cancer, confirming DDX3X as a new actionable molecular target in these tumors. Abstract Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

Over the past decades, the number of studies employing the pig brain as a model for neurochemical studies has dramatically increased. The key translational features of the pig brain are the similarities with the cortical and subcortical structures of the human brain. In addition, the caudalmost part of the pig claustrum (CL) is characterized by a wide enlargement called posterior puddle, an ideal structure for physiological recordings. Several hypotheses have been proposed for CL function, the key factor being its reciprocal connectivity with most areas of the cerebral cortex and selected subcortical structures. However, afferents from the brainstem could also be involved. The brainstem is the main source of catecholaminergic axons that play an important neuromodulatory action in different brain functions. To study a possible role of the CL in catecholaminergic pathways, we analyzed the presence and the distribution of afferents immunostained with antibodies against tyrosine hydroxylase (TH) and dopamine betahydroxylase (DBH) in the pig CL. Here we show that the CL contains significant TH immunoreactive axons contacting perikarya, whereas projections staining for DBH are very scarce. Our findings hint at the possibility that brainstem catecholaminergic afferents project to the CL, suggesting (i) a possible role of this nucleus in functions controlled by brainstem structures; and, consequently, (ii) its potential involvement in the pathophysiology of neurodegenerative pathologies, including Parkinson's disease (PD).

Involvement of the endocannabinoid system in female reproduction has been extensively described in humans with the cognate receptors and ligands being found in the ovaries and genital tract. In human, an imbalance of the endocannabinoid system is linked with both ectopic pregnancy and infertility. In bovine species anandamide levels regulate aspects of sperm-oviduct interaction. Here we report the immunohistochemical distribution of cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) in cat ovary and oviduct, using paraffin-embedded tissue samples and commercially available antibodies. We found a differential expression of both CB1R and FAAH during different stages of ovarian function and in the oviduct. CB1R was detected only in tertiary follicle granulosa cells while more immature follicles were negative. FAAH was instead found in ovarian pre-antral follicles, the oocyte cytoplasm, and in granulosa cells of primary, secondary and tertiary follicles. Secondary and tertiary follicles were also FAAH immunoreactive. Luteal cells were immunopositive for both CB1R and FAAH. Because CBR1 in oviduct was found only in ciliated cells, it might represent a specific marker at least in cats. In contrast, FAAH immunoreactivity was observed in both ciliated and non-ciliated cells. The present study may thus serve as the starting point for further investigations on the role of the endocannabinoid system in cat reproduction. Additional work will be needed to assess whether the morphological distribution of CB1R and FAAH changes in different conditions such as pre-pubertal age, follicular phase of the sexual cycle and pregnancy.

The reticular thalamic nucleus (Rt) is a sheet of neurons that surrounds the dorsal thalamus laterally, along its dorso‐ventral and rostro‐caudal axes. It consists of inhibitory neurons releasing gamma‐aminobutyric acid (GABA). This nucleus participates in the circuitry between the thalamus and the cerebral cortex, and its impairment is associated with neuro‐psychiatric disorders. In this study, we investigated the Rt anatomy of Engrailed‐2 knockout mice (En2(−/−)), a mouse model of autism spectrum disorder (ASD), using parvalbumin as an immunohistochemical marker. We compared 4‐ and 6‐week‐old wild type (WT) and En2(−/−) mice using various morphometric parameters: cell area, shape factor, circularity and cell density. Significant differences were present in 6‐week‐old male mice with different genetic background (WT vs. En2(−/−)): the Rt neurons of En2 (−/−) mice showed a bigger cell area, shape factor and circularity when compared with WT. Age (4 weeks vs. 6 weeks) influenced the shape factor of WT females, the circularity and cell density of En2 (−/−) males, and the shape factor and circularity of En2 (−/−) females. Gender affected cell density in 4‐week‐old WT mice, shape factor and cellularity of 6‐week‐old WT mice, and cell area, shape factor and cell density of En2 (−/−) at 6 weeks. Intrasubject (left–right) asymmetry of Rt was never observed. These results show for the first time that sex‐ and age‐related changes occur in the Rt GABAergic neurons of the En2 (−/−) ASD mouse model.

Despite increasing interest in the claustrum (Cl) over the last decades, its function is still a puzzling problem. Among the experimental species of potential use in Cl research, the pig is considered an interesting model, because of the similarities of its brain with the corresponding cortical and subcortical human structures. The swine Cl presents a peculiar morphology, characterized by a wide posterior enlargement, ideal for physiological investigations. There is a wealth of data on general anatomy, cytoarchitecture, and chemo architecture of the Cl, but much less is known about the dendritic morphometry of its neurons. Dendritic length and branching pattern are key features to understand the organization of the microcircuitry, and thus the delineation of the structure-function relationships of the Cl. To this effect, we undertook (a) a quantitative study of the dendrites of the spiny neurons of the swine Cl, employing the Golgi staining; and (b) an immunohistochemical analysis to describe the distribution of the parvalbumin (PV)-immunoreactive interneurons throughout the same nucleus. Taken together, the results that we report here show that the dendritic architecture and the distribution of the PV expressing interneurons change when the Cl of this species changes its shape along the rostro-caudal axis, thus suggesting a potentially specific function for the large posterior puddle. Anat Rec, 302:1638-1646, 2019. © 2019 American Association for Anatomy.

Cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) are part of the endocannabinoid system (ECB) which exerts a neuromodulatory activity on different brain functions and plays a key role in neurogenesis. Although many studies have reported FAAH and CB1R expression in the brain of different animal species, to the best of our knowledge they have never been described in the canine claustrum. Claustrum samples, obtained from necropsy of four neurologically normal dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for CB1R and FAAH. Analysis of adjacent sections incubated with the two antisera showed a complementary labeling pattern in the claustrum, with CB1R antibody staining fibers while anti-FAAH antibody stained cell bodies and the proximal portion of dendrites; this particular anatomical relationship suggests a retrograde endocannabinoid action via CB1R. CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the ECB in the dog claustrum.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances, presenting progressive neurodegeneration in the striatum and intracellular mutant Huntingtin (mHTT) aggregates in various areas of the brain. Recombinant Adeno Associated Viral (rAAV) vectors have been successfully used to transfer foreign genes to the brain of adult animals. In the present study we report a novel in vivo rat HD model obtained by stereotaxic injection of rAAV serotype2/9 containing Exon1-Q138 mHTT (Q138) and Exon1-Q17 wild type HTT (Q17; control), respectively in the right and in the left striatum, and expressed as C-terminal GFP fusions to facilitate detection of infected cells and aggregate production. Immunohistochemical analysis of brain slices from animals sacrificed twenty-one days after viral infection showed that Q138 injection resulted in robust formation of GFP-positive aggregates in the striatum, increased GFAP and microglial activation and neurodegeneration, with little evidence of any of these events in contralateral tissue infected with wild type (Q17) expressing construct. Differences in the relative metabolite concentrations (N-Acetyl Aspartate/Creatine and Myo-Inositol/Creatine) were observed by H1 MR Spectroscopy. By quantitative RT-PCR we also demonstrated that mHTT induced changes in the expression of genes previously shown to be altered in other rodent HD models. Importantly, administration of reference compounds previously shown to ameliorate the aggregation and neurodegeneration phenotypes in preclinical HD models was demonstrated to revert the mutant HTT-dependent effects in our model. In conclusion, the AAV2/9-Q138/Q17 exon 1 HTT stereotaxic injection represents a useful first-line in vivo preclinical model for studying the biology of mutant HTT exon 1 in the striatum and to provide early evidence of efficacy of therapeutic approaches.

There are age-related changes in testicular anatomy and physiology whereby there are modifications of sperm production and reproductive hormone functions. Effects of age on testicular microanatomy are well documented in humans, while there is limited understanding of these changes in dogs. The aim of this study was to evaluate age-related changes of seminiferous tubule morphology, interstitial fibrosis and spermatogenesis in dogs. Dogs (n = 32) were divided into four age groups: peripubertal (n = eight), relatively younger (n = seven), reproductively mature (n = seven) and relatively older (n = ten). Picrosirius Red stained sections were used for morphometrical analysis of testicular tissues, while the characteristics of seminiferous epithelium were assessed using a modified Johnsen scoring system for haematoxylin and eosin stained sections. Seminiferous epithelium and seminiferous tubule area increased from peripuberty to reproductive maturity, indicating there were changes during sexual maturation and subsequently there were decreases with further aging. There was a similar age-related trend for changes in seminiferous epithelium height with values being greatest in reproductively mature dogs; while there were no age-related differences in tubular diameter. Collagen content in the testicular interstitium gradually decreased from peripuberty to the age when dogs were reproductively mature and there were subsequent increases in relatively older dogs, thus, there was an association between the extent of testicular fibrosis and senescence. There was a decrease in spermatogenetic functions from relatively younger to older ages. Further investigations are warranted to establish mechanisms responsible for age-related changes of testicular morphology and related clinical implications.

Background It is commonly accepted that canine dystrophic mineralization of the hair follicle glassy membrane can be seen in hyperadrenocorticism and as a senile change in poodles. Pathology textbooks define this change as deposition of calcium salts in the form of basophilic, amorphous, granular material along collagen fibrils. Hypothesis/objectives The aim was to evaluate whether the incidence of the lesion is specific to poodles and if it is always associated with calcium deposition. Animals One hundred and forty-seven dogs divided into three groups: (i) 91 normal poodles; (ii) 40 dogs of other breeds; and (iii) 16 dogs with clinical/histopathological diagnosis of hyperadrenocorticism. Methods and materials Retrospective study; with haematoxylin and eosin, and von Kossa staining. Results Our findings demonstrate that perifollicular changes of the hair follicle glassy membrane of poodles are not always associated with calcium deposition. Conclusions and clinical importance A specific staining is needed to identify true calcium deposition when performing histological examination of the skin of poodles.

The present study aimed at setting up a standard operating histological procedure to discriminate fresh from frozen-thawed fish products of the species Merluccius merluccius (European hake). A preliminary histological analysis of fresh M. merluccius muscle was performed to select the sampling site and highlight possible time-dependent tissue alterations during shelflife. To set a suitable operational grid for discriminating the freezing process, morphological and morphometrical parameters were assessed on 90 muscle tissue samples collected from 30 fresh, 30 experimentally frozen at −20 °C and 30 Individual Quick Frozen specimens of M. merluccius. Structural score, presence of freezing vacuoles, a number of vacuoles per field higher than 1.12 and the presence of interstitial proteinaceous material, which had achieved statistical significance in group comparisons were chosen as freezing markers. Accuracy and repeatability, assessed on the analysis of two independent operators (on-training and expert), showed high analytical specificity and sensitivity and a concordant diagnostic performance regardless the operators expertise. The grid was finally validated by a single blind test on 30 additional M. merluccius commercial products and allowed the allocation of all the samples to fresh or frozen status without inconclusive results. The method could be profitably applied against fraudulent adulteration practices.

Background Cannabinoid receptors and peroxisome proliferator-activated receptor-alpha (PPAR-α) are gaining recognition as potential therapeutic targets for the treatment of skin disorders. Hypothesis/objectives The aim of this study was to investigate the distribution of cannabinoid type 1 and 2 receptors (CBR1 and CBR2) and PPAR-α in feline skin and verify whether changes occur in the course of hypersensitivity dermatitis. Animals Twelve privately owned cats. Skin samples were obtained from five healthy cats with no skin lesions and seven cats clinically diagnosed with hypersensitivity dermatitis. Methods and materials Haematoxylin and eosin stained skin sections were investigated for histopathological changes. Indirect immunofluorescence for CBR1, CBR2 and PPAR-α was performed on paraffin-embedded sections, and antibody specificity tested by Western blot analysis. Results Skin samples from cats with hypersensitivity dermatitis were all histopathologically diagnosed with eosinophilic dermatitis. CB receptors and PPAR-α were distributed throughout the skin in both healthy and allergic cats. In normal feline skin, these receptors were mainly distributed in the epithelial compartment. Receptor expression increased in hypersensitivity compared to healthy skin, with the main distribution changes being suprabasal for CBR1, dermal for CBR2 and marked expression of PPAR-α in hyperplastic epidermis and perivascular infiltrate. Conclusions and clinical importance Increased expression of cannabinoid receptors in the skin of cats with hypersensitivity dermatitis suggests an endogenous protective strategy and may support the use of natural cannabinoid receptor or PPAR-α agonists to treat feline hypersensitivity dermatitis.

Summary Reasons for performing study Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. Objectives To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. Study design Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. Methods Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. Results Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. Conclusions Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man.

The aim of this study was to investigate the ability of suturable platelet-rich plasma (PRP) membrane to promote peripheral nerve regeneration after neurotmesis and neurorraphy.A total of 36 rats were used: 32 animals underwent surgery and were split in two groups. An interim sacrifice was performed at 6 weeks postsurgery and final sacrifice at 12 weeks; four animals did not sustain nerve injury and served as control. Clinical, electromyographic (EMG), gross, and histological changes were assessed. The EMG signal was evaluated for its amplitude and frequency spectrum. Number of regenerating fibers, their diameter, and myelin thickness were histologically analyzed.Both EMG parameters showed a significant (p 0.05) effect of treatment at 6 and 12 weeks postsurgery. At 6 weeks, the fiber density was statistically different between treated and untreated animals with a higher observed density in treated nerves. No difference in fiber density was observed at 12 weeks postsurgery. The distribution of fiber diameters showed an effect at 12 weeks when only the sections of the nerves sutured with PRP showed fibers with diameters greater than 6 µm.Our data show that the application of a PRP fibrin membrane around the neurorraphy improves the nerve regeneration process in a rat sciatic nerve model. The use of PRP as a suturable membrane could perform an action not only as a source of bioactive proteins but also as a nerve guide to hold the scar reaction and thus improve axonal regeneration.

The re-epithelialization process in equine wound healing is incompletely described. For epithelial cells to migrate during embryogenesis they undergo epithelial-to-mesenchymal transition (EMT); this phenotypic transition occurs during wound healing in humans and rodents, but it has not been investigated in horses.To investigate keratinocyte differentiation and EMT in equine experimental excisional limb and body wounds healing by second intention.Six adult research horses.Immunohistochemical analysis was used to detect expression of the differentiation markers cytokeratin (CK)10, CK14, loricrin and peroxisome proliferator-activated receptor alpha (PPAR-α), and of the EMT markers E-cadherin and N-cadherin in normal limb and body skin, and biopsies from limb and body wounds.Loricrin and CK10 were expressed in normal skin and periwound skin but not in migrating epithelium of body and limb wounds. However, they reappeared at the migrating epithelial tip of body wounds only. CK14 and PPAR-α had uniform distribution throughout the migrating epithelium. N-cadherin was not expressed in normal unwounded skin but was detected in periwound skin adjacent to the wound margin. E-cadherin expression decreased at the wound margin.Presence of N-cadherin suggests that cadherin switching occurred during wound healing, this may be an indication that EMT occurs in horses. To the best of the authors' knowledge, this has never been described in horses before and warrants further investigation to assess the clinical implications. The tip of the migrating epithelium in body wounds appeared more differentiated than limb wounds, which could be part of the explanation for the superior healing of body wounds.Le processus de re-épithélialisation dans la cicatrisation des plaies chez le cheval n'est pas entièrement décrit. Pour que les cellules épithéliales migrent au cours de l'embryogénèse, elles passent par une transition épithéliale-mésenchymateuse (EMT); cette transition phénotypique se produit au cours de la cicatrisation chez l'homme et les rongeurs mais n'a pas été étudiée chez le cheval. HYPOTHÈSES/OBJECTIFS: Etudier la différenciation kératinocytaire et l’EMT au cours de plaies expérimentales et la cicatrisation par seconde intention chez le cheval.Six chevaux de recherche adultes. MATÉRIEL ET MÉTHODE: Une analyse immunohistochimique a été utilisée pour détecter l'expression des marqueurs de différenciation : cytokératines (CK)10, CK14, loricrine et PPAR-α (peroxisome proliferator-activated receptor alpha), les marqueurs d’EMT : E-cadhérine et N-cadhérine dans la peau normale d'un membre et du tronc et des biopsies des plaies des membres et du tronc. RÉSULTATS: La loricrine et CK10 étaient exprimées dans la peau normale et en périphérie des plaies cutanées mais par dans l’épithélium en migration des plaies du tronc et des membres. Cependant, elles réapparaissent à l'extrémité de l’épithélium en migration dans les plaies du tronc seulement. CK14 et PPAR-α ont une distribution uniforme au sein de l’épithélium migrant. La N-cadhérine n’était pas exprimée dans la peau normale intacte mais était détectée en périphérie des plaies cutanées adjacentes aux marges des plaies. L'expression d'E-cadhérine a diminué en marge des plaies.La présence de N-cadhérine suggère qu'une transformation se produit au couts de la cicatrisation, ce qui peut être une indication que l’EMT de produit chez le cheval. A la connaissance des auteurs, ceci n'a jamais été décrit chez le cheval et nécessite d'autres études pour déterminer les implications clinques. L'extrémité de l’épithélium en migration dans les plaies corporelles semble plus différenciée que pour les plaies des membres, ce qui pourrait être une partie de l'explication de la meilleure capacité de cicatrisation des plaies tronculaires.INTRODUCCIÓN: el proceso de reepitelización en la cicatrización de heridas equinas se ha descrito de forma incompleta. Para que las células epiteliales migren durante la embriogénesis, sufren una transición epitelial a mesenquimatosa (EMT); esta transición fenotípica ocurre durante la cicatrización de heridas en humanos y roedores, pero no se ha investigado en caballos. HIPÓTESIS/OBJETIVOS: investigar la diferenciación de queratinocitos y la EMT en la extremidad y el cuerpo tras escisión experimental creando heridas que cicatrizaron por segunda intención. ANIMALES: Seis caballos adultos de investigación. MÉTODOS Y MATERIALES: se utilizó análisis inmunohistoquímico para detectar la expresión de los marcadores de diferenciación citoqueratina (CK) 10, CK14, loricrina y el receptor activado alfa proliferador de peroxisomas (PPAR-α) y de los marcadores de EMT E-cadherin y N-cadherin en piel normal de extremidades y cuerpo, y biopsias de heridas de las mismas zonas. RESULTADOS: loricrina y CK10 se expresaron en la piel normal y en la piel cercana a la herida, pero no en el epitelio migratorio de las heridas del cuerpo y las extremidades. Sin embargo, reaparecieron solo en el punto de avance epitelial migratorio de las heridas corporales. CK14 y PPAR- α tuvieron una distribución uniforme en todo el epitelio migratorio. La N-cadherina no se expresó en la piel normal sin herida, pero se detectó en la piel adyacente al margen de la herida. La expresión de E-cadherina disminuyó en el margen de la herida. CONCLUSIONES E IMPORTANCIA CLÍNICA: la presencia de N-cadherina sugiere que se produjo un cambio de cadherina durante la cicatrización de la herida, lo que puede ser una indicación de que se produce EMT en caballos. A entender de los autores, este fenómeno no había sido descrito en caballos y merece más investigación para evaluar las implicaciones clínicas. El extremo de avance del epitelio migratorio en las heridas del cuerpo parecía más diferenciado que en las heridas de las extremidades, lo que podría explicar en parte la mejor cicatrización de las heridas corporales.Der Prozess der Reepithelialisierung bei der Wundheilung von Pferden ist nur unvollständig beschrieben. Damit Epithelzellen während der Embryogenese wandern können, machen sie eine Transformation von epithelial-zu-mesenchymal (EMT) durch; diese phänotypische Transition erfolgt während der Wundheilung beim Menschen und bei Nagern, ist aber bei Pferden noch nicht untersucht worden.Eine Untersuchung der Keratinozytendifferenzierung und EMT beim Pferd bei experimentellen exzisionalen Extremitäten und Körperwunden mit Sekundärheilung.Sechs adulte Versuchspferde.Es wurde eine immunhistochemische Analyse durchgeführt, um die Exprimierung der Differenzierungsmarker Zytokeratin (CK)10, CK14, Loricrin und Peroxisome Proliferator-aktivated Receptor alpha (PPAR-α), sowie der EMT Marker E-Cadherin und N-Cadherin in normaler Haut von Extremitäten und Körper, sowie Biopsien von Extremitäten und Körperwunden festzustellen.Loricrin und CK10 wurden in normaler Haut und in Haut rundum den Wunden exprimiert, aber nicht im migrierenden Epithel von Wunden an Körper und Extremitäten. Sie tauchten jedoch nur an der migrierenden Epithelspitze von Körperwunden wieder auf. CK14 und PPAR-a zeigten eine einheitliche Verteilung im migrierenden Epithel. N-Cadherin wurde in normaler nicht verwundeter Haut nicht exprimiert, wurde jedoch in Haut rundum die Wunden im Anschluss an den Wundrand gefunden. Die Exprimierung von E-Cadherin nahm Richtung Wundrand ab.Das Auftreten von N-Cadherin weist darauf hin, dass Cadherin Switching während der Wundheilung auftritt, was ein Indikator dafür sein kann, dass EMT bei Pferden auftritt. Nach bestem Wissen der Autoren wurde das bei Pferden bisher nicht beschrieben und macht weitere Untersuchungen nötig, um die klinischen Implikationen zu erfassen. Die Spitze des migrierenden Epithels in den Körperwunden zeigte sich besser ausdifferenziert als Wunden an den Extremitäten, was teilweise die bessere Wundheilung von Körperwunden erklären könnte.背景: 馬の創傷治癒における上皮再形成過程における記述は不完全である。胚形成中に上皮細胞が遊走するため、それらは上皮間葉転換(EMT)を受ける。この表現型の移行は、人およびげっ歯類の創傷治癒中に起こるが、馬では調べられていない。 仮説/目的: 本研究の目的は、馬の実験的切除肢および第二の目的として体幹創傷治癒過程におけるケラチノサイト分化およびEMTを調査することである。 被験動物: 6頭の成体研究用馬。 材料および方法: 免疫組織化学的解析を使用して、正常な四肢および体幹、そして四肢および体幹の創傷から生検し、皮膚における分化マーカーであるサイトケラチン(CK)10、CK14、ロリクリンおよびペルオキシソーム増殖因子活性化受容体α(PPAR -α)、ならびにEMTマーカーであるE-カドヘリンおよびN-カドヘリンの発現を検出した。 結果: ロリクリンおよびCK10は、正常皮膚および創傷部周囲の皮膚において発現したが、体幹部および四肢創傷部の遊走上皮では発現しなかった。しかしながら、それらは、体幹創傷部における遊走上皮先端にのみ再出現した。 CK14およびPPAR-αは、遊走上皮全体に均一に分布していた。 N-カドヘリンは、正常な創傷のない皮膚では発現しなかったが、創傷縁に隣接した創傷周囲の皮膚において検出された。 E-カドヘリン発現は創傷縁で減少した。 結論と臨床的重要性: N-カドヘリンの存在は、カドヘリンの切り替えが創傷治癒中に起こったことを示唆しており、これはEMTが馬において起こることの指標となる可能性がある。著者の知る限りでは、これまで馬に記載されたことがなく、臨床的意義を評価するためのさらなる調査が必要である。体幹創傷部における移動上皮の先端は、四肢創傷部と比較して分化しているように見えた。これは、体幹創傷部の優れた治癒能力に対する説明の一部となる可能性がある。.背景: 马伤口愈合中的上皮再生过程尚未被充分报道。上皮细胞在胚胎发生过程中发生上皮向间质转化(EMT); 这种表型转变发生在人类和啮齿动物的伤口愈合期,但尚未对马进行过相关研究。 假设/目标: 通过实验性切开马的肢体和躯体伤口制造二期愈合,研究角质细胞的分化和EMT。 动物: 六匹成年实验马。 方法和材料: 免疫组化分析检测分化标志物细胞角质(CK)10、CK14、兜甲蛋白、过氧化物酶体增殖物激活受体α(PPAR-α)的表达;检测EMT标记物E-钙黏蛋白和N-钙黏蛋白在正常肢体和躯体皮肤中的表达,并对肢体和躯体伤口进行活组织检查。 结果: 兜甲蛋白和CK10在正常皮肤和创周皮肤中表达,但在躯体和肢体伤口的移行上皮中不表达。然而,它们又会出现在躯体伤口的移行上皮顶端。CK14和PPAR-a在移行上皮中均匀分布。 N-钙黏蛋白在正常未受伤的皮肤中不表达,但可在伤口边缘附近的创周皮肤中被检测到。E-钙黏表达在伤口边缘减少。 结论和临床意义: N-钙黏蛋白的存在表明,在伤口愈合过程中发生了钙黏蛋白转换,这表明EMT可发生在马身上。据作者所知,此前从未有马的相关报道,需要进一步调查以评估临床意义。 躯体创面移行上皮顶端的分化程度明显高于肢体创面,这可能是躯体创面愈合较好的原因之一。.O processo de reepitelização na cicatrização de feridas de equinos é incompletamente descrito. Para que as células epiteliais migrem durante a embriogênese, elas passam por transição epitelial-mesenquimal (EMT); esta transição fenotípica ocorre durante o processo de cicatrização em humanos e roedores, mas não foi ainda investigado em equinos. HIPÓTESE/OBJETIVOS: Investigar a diferenciação de queratinócitos em EMT amostras excisionais de feridas cicatrizando por segunda intenção no corpo e membros de equinos.Seis cavalos adultos de experimentação. MÉTODOS E MATERIAIS: Utilizou-se análise imunohistoquímica para detectar a expressão dos marcadores de diferenciação citoqueratina (CK)10, CK14, loricrina e receptor alfa ativado por proliferador peroxissomo (PPAR-α), e dos marcadores de EMT E-caderina e N-caderina na pele normal de membros e corpo, e biópsias de feridas no corpo e membros.A loricrina e a CK10 estavam sendo expressadas na pele normal e perilesional, mas não no epitélio migrante das feridas de corpo e membros. Entretanto, elas reapareceram apenas no epitélio migrante na ponta das feridas do corpo. CK14 e PPAR-α apresentaram uma distribuição uniforme ao longo do epitélio migrante. A N-caderina não foi expressada na pele sem ferida, mas foi detectada na pele perilesional adjacente à margem da ferida. A expressão de E-caderina decresceu na margem da ferida. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: A presença de N-caderina sugere que houve troca de caderina ocorreu durante o processo de cicatrização, isto pode ser um indicador de que EMT ocorre em equinos. De acordo com os conhecimentos do autor, isso nunca havia sido descrito em cavalos antes e as feridas no corpo pareceram ser mais diferenciadas que as feridas nos membros, o que pode ser uma parte da explicação da superioridade da cicatrização em feridas corporais.

Background Hair cycle arrest (alopecia X) refers to a canine alopecic condition of unknown pathogenesis, characterized by symmetrical, nonpruritic and noninflammatory alopecia that spares the head and distal extremities. Hypothesis/Objectives The objective of this study was prospectively to evaluate the efficacy of a 4.7 mg deslorelin implant in the treatment of intact male and neutered female dogs affected by hair cycle arrest. Animals Ten Pomeranian dogs (eight intact males, two neutered females), four Italian spitz (three intact males, one neutered female), three miniature poodles (two intact males, one neutered female), two Siberian huskies (both intact males) and one intact male chow chow dog with confirmed hair cycle arrest were included in the study. Methods Each dog was treated with a subcutaneous sterile implant containing 4.7 mg deslorelin. Responder dogs were re-implanted 6 months after the first implant in order to obtain a 1 year pharmacological exposure and 1 year of follow-up. Results Hair regrowth was visible within 3 months in 12 of 16 intact male dogs (75%); no hair regrowth was noted in any neutered female dogs. The overall response to therapy was 60%. No adverse effects were noted. Conclusions and clinical importance These findings suggest that deslorelin may be a treatment option for intact male dogs with idiopathic hair cycle arrest. Deslorelin is an alternative to current therapies and castration.

One of the most challenging requirements of a successful bone tissue engineering approach is the development of scaffolds specifically tailored to individual tissue defects. Besides materials chemistry, well-defined scaffold’s structural features at the micro- and macro-levels are needed for optimal bone in-growth. In this study, polymeric fibrous scaffolds with a controlled internal network of pores and modelled on the anatomical shape and dimensions of a critical size bone defect in a rabbit’s radius model were developed by employing a computer-aided wet-spinning technique. The tailored scaffolds made of star poly(ε-caprolactone) or star poly(ε-caprolactone)–hydroxyapatite composite material were implanted into 20-mm segmental defects created in radial diaphysis of New Zealand white rabbits. Bone regeneration and tissue response were assessed by X-rays and histological analysis at 4, 8 and 12 weeks after surgery. No signs of macroscopic and microscopic inflammatory reactions were detected, and the developed scaffolds showed a good ability to support and promote the bone regeneration process. However, no significant differences in osteoconductivity were observed between star poly(ε-caprolactone) and star poly(ε-caprolactone)–hydroxyapatite scaffolds. Long-term study on implanted star poly(ε-caprolactone) scaffolds confirmed the presence of signs of bone regeneration and remodelling, particularly evident at 24 weeks.

Full-thickness canine skin organ culture models could provide an entirely new opportunity for studying wound healing, keratinization disorders and allergic skin diseases, all of which have high prevalence and severe impact on canine quality of life. Here we present a canine organ culture method for the short-term maintenance of full-thickness, adult, canine skin in serum-free medium and investigate the possibility to induce mast cell degranulation ex vivo. General morphological features were maintained up to day 7. Epidermal thickness started to decrease from day 4 of culture. No changes were observed in epidermal pigmentation. Keratinocyte proliferation started to significantly decrease at day 7. Immunostaining for cytokeratin 10, cytokeratin 14 and loricrin was evident from day 1 to day 7. Compound 48/80 induced mast cell degranulation. This was the first attempt to establish a dog skin organ culture and document good preservation of most cutaneous structures till day 7. This method may help in dissecting canine skin biology in physiological and pathological conditions and to study drug mechanism of action in a biologically relevant environment.

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.

Although canine skin models are already available as either monocellular or organotypic cultures, they only partly recapitulate normal skin morphological features and function. The objective of this study was to establish a canine serum-free skin organ culture model and verify whether dexamethasone could rescue epidermal growth factor-induced changes. The study of morphological changes as a response to pharmacological substances may indeed help to investigate skin physiology and pathology. Normal skin was obtained from five client-owned dogs subjected to surgical procedures unrelated to dermatological conditions. Two experimental designs were performed: (i) two-week viability of the skin culture; (ii) dexamethasone (DMS) inhibition of epidermal growth factor (EGF)-induced effects. Serum-free submerged organ cultures were established in Williams' E medium supplemented with penicillin-streptomycin, insulin, hydrocortisone and l-glutamine. General morphological features of skin anatomical structures were well maintained up to day 14, scattered pyknotic nuclei were visible in the epidermis from day 7. Normal keratinocyte differentiation was confirmed by cytokeratin (K) 10, K14 and loricrin immunostaining. Epidermal thickness did not decrease throughout the study. A decrease in keratinocyte proliferation was observed at day 7 and 14. Treatment with EGF induced both keratinocyte proliferation and thickening of the epidermis; both responses were counteracted by DMS. Treatment with EGF increased the length of epithelial tongues at the edge of the skin explants; this effect was further enhanced by DMS supplementation. Our findings demonstrate the potential use of a full-thickness canine skin organ culture model for the study of skin physiology and pharmacological response to exogenous compounds, especially in the field of re-epithelialisation and keratinization disorders.

Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expression was studied in unwounded skin and in samples from 1, 2, 3, 4, and 6 wk old wounds of the body and limb. Temporal gene expression was determined by semiquantitative RT-PCR, while protein expression was mapped immunohistochemically. The temporal pattern of expression for both genes was similar; wounding caused immediate upregulation of mRNA, which did not return to baseline by the end of the study, and overexpression was noted in body relative to limb wounds. Immunostaining for THBS2 and SPARC was induced by wounding, though no differences in stain location or intensity were detected between body and limb wounds. This study is the first to characterize equine cDNA for THBS2 and SPARC and to document mRNA expression over the different phases of repair. THBS2 and SPARC might modulate angiogenesis during wound healing in the horse, which could protect against the disproportionate fibroplasia commonly afflicting limb wounds and leading to the development of exuberant granulation tissue.