Anatomical distribution of spontaneous iron pigment overload in the liver of han wistar and sprague-dawley rats

The authors performed a retrospective study to determine and describe the incidence of spontaneous pigment overload in the liver of control Han Wistar and Sprague-Dawley rats. Data was collected from 1170 control animals (550 Han-Wistar and 620 Sprague-Dawley) from control dose groups from long term regulatory studies (104-week carcinogenicity studies). Further 628 control animals (300 Han-Wistar and 328 Sprague-Dawley) from control dose groups from short term regulatory studies (13-week and 4 weeks studies) evaluated at the authors’ laboratory between 2009 and 2011. Livers from Han Wistar and Sprague-Dawley rats were re-evaluated using special stains to identify the nature of the pigments. In the periportal hepatocytes and in scattered sinusoidal Kupffer cells, the predominant pigment was identified as haemosiderin and a diagnosis of spontaneous iron overload was made. A comparison between the two strains revealed higher incidences of iron overload in Han Wistar rats than Sprague-Dawley rats. A significant sex difference was observed in both strains but was greater in Han Wistar rats. An age-related increase in the incidence and severity of pigment deposition was also apparent. Since there is little compiled data on spontaneous pigment overload in the liver the aim of this report was to summarize and discuss the incidence, distribution and factors affecting the occurrence of this background finding in control rats on toxicity studies

Preferential epithelial expression of type-1 cannabinoid receptor (CB1R) in the developing canine embryo

The use of cannabinoid receptor agonists is gaining a strong interest both in human and veterinary medicine. The potential use of cannabimimetic compounds in companion animals was reviewed in 2007 for their role in tissue inflammation and pain. A better knowledge of type-1 cannabinoid receptor (CB1R) expression on the target population may help in risk management in order to prevent unwanted side effects. We used 30-days old canine embryos to describe the distribution of CB1R by means of immunohistochemistry with a commercially available antibody.CB1R immunoreactivity was mainly epithelial and included most structures of central and peripheral nervous system, inner ear, olfactory epithelium and related structures, eye and thyroid. Further investigative research on the role of the endocannabinoid system in the developmental biology field is needed, however, we show that in the canine species we must consider pregnancy as risk condition for developmental abnormalities that may arise upon the use of CB1R receptor agonists

Cannabinoid receptor types 1 and 2 and peroxisome proliferator-activated receptor-α: distribution in the skin of clinically healthy cats and cats with hypersensitivity dermatitis

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

Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair

<p>Abstract</p> <p>Background</p> <p>Wound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to <it>CTNNB1 </it>(coding for β-catenin) and <it>PECAM1</it>, genes potentially contributing to the proliferative phase of repair, were previously identified in a mRNA expression study as being up-regulated in 7 day wound biopsies from horses. The aim of the present study was to clone full-length equine <it>CTNNB1 </it>and <it>PECAM1 </it>cDNAs and to study the spatio-temporal expression of mRNAs and corresponding proteins during repair of body and limb wounds in a horse model.</p> <p>Results</p> <p>The temporal pattern of the two genes was similar; except for <it>CTNNB1 </it>in limb wounds, wounding caused up-regulation of mRNA which did not return to baseline by the end of the study. Relative over-expression of both <it>CTNNB1 </it>and <it>PECAM1 </it>mRNA was noted in body wounds compared to limb wounds. Immunostaining for both β-catenin and PECAM1 was principally observed in endothelial cells and fibroblasts and was especially pronounced in wounds having developed exuberant granulation tissue.</p> <p>Conclusion</p> <p>This study is the first to characterize equine cDNA for <it>CTNNB1 </it>and <it>PECAM1 </it>and to document that these genes are expressed during wound repair in horses. It appears that β-catenin may be regulated in a post-transcriptional manner while PECAM1 might help thoracic wounds mount an efficient inflammatory response in contrast to what is observed in limb wounds. Furthermore, data from this study suggest that β-catenin and PECAM1 might interact to modulate endothelial cell and fibroblast proliferation during wound repair in the horse.</p

Osteopontin expression in healing wounds of horses and in human keloids

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

Increased levels of palmitoylethanolamide and other bioactive lipid mediators and enhanced local mast cell proliferation in canine atopic dermatitis

Background: Despite the precise pathogenesis of atopic dermatitis (AD) is unknown, an immune dysregulation that causes Th2-predominant inflammation and an intrinsic defect in skin barrier function are currently the two major hypotheses, according to the so-called outside-inside-outside model. Mast cells (MCs) are involved in AD both by releasing Th2 polarizing cytokines and generating pruritus symptoms through release of histamine and tryptase. A link between MCs and skin barrier defects was recently uncovered, with histamine being found to profoundly contribute to the skin barrier defects. Palmitoylethanolamide and related lipid mediators are endogenous bioactive compounds, considered to play a protective homeostatic role in many tissues: evidence collected so far shows that the anti-inflammatory effect of palmitoylethanolamide depends on the down-modulation of MC degranulation. Based on this background, the purpose of the present study was twofold: (a) to determine if the endogenous levels of palmitoylethanolamide and other bioactive lipid mediators are changed in the skin of AD dogs compared to healthy animals; (b) to examine if MC number is increased in the skin of AD dogs and, if so, whether it depends on MC in-situ proliferation. Results: The amount of lipid extract expressed as percent of biopsy tissue weight was significantly reduced in AD skin while the levels of all analyzed bioactive lipid mediators were significantly elevated, with palmitoylethanolamide showing the highest increase. In dogs with AD, the number of MCs was significantly increased in both the subepidermal and the perifollicular compartments and their granule content was significantly decreased in the latter. Also, in situ proliferation of MCs was documented. Conclusions: The levels of palmitoylethanolamide and other bioactive lipid mediators were shown to increase in AD skin compared to healthy samples, leading to the hypothesis that they may be part of the body's innate mechanisms to maintain cellular homeostasis when faced with AD-related inflammation. In particular, the increase may be considered a temptative response to down-regulating the observed elevation in the number, functionality and proliferative state of MCs in the skin of AD dogs. Further studies are warranted to confirm the hypothesis

Development of a short-term canine full-thickness skin organ culture method under serum-free conditions

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

Proteomic Profiling Reveals Specific Molecular Hallmarks of the Pig Claustrum

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

Recombinant adeno associated viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery

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