Proof of concept of a new autologous skin substitute for the treatment of deep wounds in dogs.

Autologous skin grafts are effective for the repair of large skin wounds, but the availability of large amounts of skin is often limited. Through bioengineering, several autologous skin substitutes have been developed for use in human clinical practice. However, few skin substitutes are available for use in animals. The aim of this study was to develop and assess an engineered autologous skin substitute for the treatment of deep wounds in veterinary medicine. Canine keratinocytes and fibroblasts were isolated after double enzyme digestion from 8mm punch biopsies from four healthy Beagle dogs. Skin substitutes were constructed on a fibrin-based matrix and grafting capacity was assessed by xenografting in six athymic mice. Bioengineered autologous skin was assessed clinically in two dogs with large deep skin wounds. The canine skin construct was ready for use within 12-14days after the initial biopsy specimens were obtained. Grafting capacity in this model was confirmed by successful grafting of the construct in athymic mice. In both dogs, grafts were established and permanent epithelialisation occurred. Histological studies confirmed successful grafting. This full thickness skin substitute developed for the management of large skin defects in dogs appears to be a safe and useful tool for clinical veterinary practice. Further studies are needed to validate its efficacy for the treatment of deep wounds.

Spontaneously Arising Canine Glioma as a Potential Model for Human Glioma.

Human gliomas are malignant brain tumours that carry a poor prognosis and are composed of a heterogeneous population of cells. There is a paucity of animal models available for study of these tumours and most have been created by genetic modification. Spontaneously arising canine gliomas may provide a model for the characterization of the human tumours. The present study shows that canine gliomas form a range of immunohistochemical patterns that are similar to those described for human gliomas. The in-vitro sphere assay was used to analyze the expansion and differentiation potential of glioma cells taken from the periphery and centre of canine tumours. Samples from the subventricular zone (SVZ) and contralateral parenchyma were used as positive and negative controls, respectively. The expansion potential for all of these samples was low and cells from only three cultures were expanded for six passages. These three cultures were derived from high-grade gliomas and the cells had been cryopreserved. Most of the cells obtained from the centre of the tumours formed spheres and were expanded, in contrast to samples taken from the periphery of the tumours. Spheres were also formed and expanded from two areas of apparently unaffected brain parenchyma. The neurogenic SVZ contralateral samples also contained progenitor proliferating cells, since all of them were expanded for three to five passages. Differentiation analysis showed that all cultured spheres were multipotential and able to differentiate towards both neurons and glial cells. Spontaneously arising canine gliomas might therefore constitute an animal model for further characterization of these tumours.

Cartilage resurfacing potential of PLGA scaffolds loaded with autologous cells from cartilage, fat, and bone marrow in an ovine model of osteochondral focal defect.

Current developments in tissue engineering strategies for articular cartilage regeneration focus on the design of supportive three-dimensional scaffolds and their use in combination with cells from different sources. The challenge of translating initial successes in small laboratory animals into the clinics involves pilot studies in large animal models, where safety and efficacy should be investigated during prolonged follow-up periods. Here we present, in a single study, the long-term (up to 1 year) effect of biocompatible porous scaffolds non-seeded and seeded with fresh ex vivo expanded autologous progenitor cells that were derived from three different cell sources [cartilage, fat and bone marrow (BM)] in order to evaluate their advantages as cartilage resurfacing agents. An ovine model of critical size osteochondral focal defect was used and the test items were implanted arthroscopically into the knees. Evidence of regeneration of hyaline quality tissue was observed at 6 and 12 months post-treatment with variable success depending on the cell source. Cartilage and BM-derived mesenchymal stromal cells (MSC), but not those derived from fat, resulted in the best quality of new cartilage, as judged qualitatively by magnetic resonance imaging and macroscopic assessment, and by histological quantitative scores. Given the limitations in sourcing cartilage tissue and the risk of donor site morbidity, BM emerges as a preferential source of MSC for novel cartilage resurfacing therapies of osteochondral defects using copolymeric poly-D,L-lactide-co-glycolide scaffolds.

Transitory improvement of articular cartilage characteristics after implantation of polylactide:polyglycolic acid (PLGA) scaffolds seeded with autologous mesenchymal stromal cells in a sheep model of critical-sized chondral defect.

Clinical translation of emerging technologies aiming at cartilage resurfacing is hindered by neither the appropriate scaffold design nor the optimal cell source having been defined. Here, critical-sized, chondral-only focal defects were created in sheep and treated with clinical-grade, co-polymeric poly-lactide:polyglycolic acid scaffolds either alone or seeded with 3.3 × 10(6) ± 0.4 × 10(6) autologous bone marrow-derived mesenchymal stromal cells and studied over 12 month follow-up. An untreated group was included for comparison. Second-look arthroscopy performed at 4 months post-treatment evidenced the generation of neocartilage of better quality in those defects treated with cells. However, macroscopic scores in the cell-treated group declined significantly from 7.5 ± 2.3 at 4 months to 3.1 ± 2.6 (p = 0.0098) at 12 months post-treatment, whereas the other two experimental groups remained unaltered during 4-12 month post-treatment. The effectiveness of the cell-based approach proposed in this study is thus restricted to between months 1 and 4 post-treatment.

Mapping of neurotrophins and their receptors in the adult mouse brain and their role in the pathogenesis of a transgenic murine model of bovine spongiform encephalopathy.

Neurotrophins are a family of growth factors that act on neuronal cells. The neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3, -4 and -5. The action of neurotrophins depends on two transmembrane-receptor signalling systems: (1) the tropomyosin-related kinase (Trk) family of tyrosine kinase receptors (Trk A, Trk B and Trk C) and (2) the p75 neurotrophin receptor (p75(NTR)). The interaction between neurotrophic factors and their receptors may be involved in the mechanisms that regulate the differential susceptibility of neuronal populations in neurodegenerative diseases. The aim of the present study was to evaluate the role of neurotrophins in the pathogenesis of bovine spongiform encephalopathy (BSE) using a transgenic mouse overexpressing bovine prnp (BoTg 110). Histochemistry for Lycopersicum esculentum agglutinin, haematoxylin and eosin staining and immunohistochemistry for the abnormal isoform of the prion protein (PrP(d)), glial fibrillary acidic protein (GFAP), NGF, BDNF, NT-3 and the receptors Trk A, Trk B, Trk C and p75(NTR) was performed. The lesions and the immunolabelling patterns were assessed semiquantitatively in different areas of the brain. No significant differences in the immunolabelling of neurotrophins and their receptors were observed between BSE-inoculated and control animals, except for p75(NTR), which showed increased expression correlating with the distribution of lesions, PrP(d) deposition and gliosis in the BSE-inoculated mice.

Influence of seminal plasma on leucocyte migration and amount of COX-2 protein in the jenny endometrium after insemination with frozen-thawed semen.

After mating, seminal plasma has an immuno-modulatory effect on the endometrium in some mammals. In jennies, achieving conception via artificial insemination (AI) with frozen-thawed semen is generally much more difficult than in mares. The endometrial inflammatory response is hypothesized to be a contributing factor to the lesser fertility. Following a cross-over experimental design, the uterine inflammatory response of six jennies was evaluated at 6h after AI with frozen-thawed semen (deposited in the uterine body) in the presence or absence of autologous seminal plasma (+SP or -SP). The endometrial cytology and histology of the animals were examined by uterine lavage, uterine swabbing and biopsy. The amount of cyclooxygenase-2 (COX-2) protein in endometrial cells was also evaluated. As a control (C), the same examinations were made before any AI procedure (i.e., when the jennies were in oestrus). Large numbers of polymorphonuclear neutrophils (PMN) were observed in the -SP and +SP cytology and biopsy samples; more than in the C samples. The -SP samples also had intense COX-2 labelling; less labelling was detected in the +SP and C samples (no significant difference between these latter two types). Thus, while the presence of SP does not change the post-AI number of PMNs with regard to that detected in its absence, it does reduce COX-2 protein. Further research into the complex mix of molecules in SP and its effects during AI might help increase the pregnancy rates achieved in jennies.

Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen.

Artificial insemination (AI) involving the placing of frozen-thawed semen directly into the jenny uterine body is associated with very low pregnancy rates. This might be because of an exacerbation of the acute response of the endometrium to sperm, as seen in mares with persistent induced mating endometritis. Pregnancy rates can be increased in such mares, however, by including anti-inflammatory treatments in the insemination protocol (Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. Theriogenology 2008;70:1093-100; Rojer H, Aurich C. Treatment of persistent mating-induced endometritis in mares with the non-steroid anti-inflammatory drug vedaprofen. Reprod Domest Anim 2010;45:e458-60). To investigate the endometritis caused by the use of frozen-thawed semen in jennies, and to assess the response to ketoprofen treatment, endometrial cytological samples and biopsies from six healthy jennies were examined in a crossover design experiment. Samples were taken from jennies in estrus (E; control) and at 6 hours after AI with or without ketoprofen (+K and -K, respectively). Ketoprofen was administered iv 24 hours before and for 4 days after insemination (total = 2.2 mg/kg/24 hours for 5 days). All animals showed a severe inflammatory response to semen deposition. Polymorphonuclear neutrophil numbers in the cytological smears and biopsies differed significantly between the +K and E animals. No significant differences were recorded, however, between the +K and -K treatments. Eosinophils were observed in all sample types from all groups; these cells appear to be a feature of the normal jenny endometrium. Slight fibrosis was observed in some biopsies, but no significant relationship with inflammation was found. Intense cyclooxygenase-2 (COX-2) immunohistochemical labeling was detected in the -K biopsies. Less intense labeling was seen in those of the +K animals, and mainly localized in the stratum compactum. No differences in COX-2 labeling were observed between the +K and E animals. Plasma concentrations of ketoprofen remained detectable until 2 hours after administration, after which the compound was rapidly eliminated. In summary, jennies are susceptible to endometritis after insemination with frozen-thawed semen. Ketoprofen reduces this inflammation by inhibiting COX-2; no reduction in the number of polymorphonuclear neutrophils occurs. The physiological and pharmacological characteristics of jennies should be taken into account when designing treatments for acute endometritis aimed at enhancing pregnancy rates after insemination with frozen-thawed sperm.

Late stage cathepsin C, CXCL13 and Ki-67 overexpression correlate with regional neuropathology in a BSE transgenic murine model.

A DNA microarray-based gene expression analysis study was performed with bovine spongiform encephalopathy (BSE) transgenic mice. Several genes were found to be overexpressed including the lysosomal enzyme cathepsin C, the chemokine CXCL13 and a number of genes related to cellular proliferation. The brains from terminal stage, BSE inoculated, 'bovinized', transgenic mice were subjected to immunohistochemistry with antibodies against these two proteins and Ki-67, a cell proliferation marker, to assess the biological relevance of the gene expression changes. Differential expression of cathepsin C and CXCL13 proteins and increased expression of Ki-67 was observed. These changes were localized to areas of deposition of PrP(res) and spongiform change and to areas showing an astroglial and microglial response. These findings suggest that these proteins are involved in the mechanisms leading to the establishment of transmissible spongiform encephalopathy.

Expression of KIT receptor in feline cutaneous mast cell tumors.

Twenty-seven feline cutaneous mast cell tumors (MCTs) were selected for this retrospective study. Samples were routinely processed and stained with hematoxylin and eosin (HE) and toluidine blue, and tumors were classified as well-differentiated (19/27), atypical or poorly granulate (7/27), and pleomorphic (1/27). Immunohistochemistry to detect KIT protein was performed on all samples. The immunoreactivity was recorded by distribution within the tumor, cellular location, and intensity. Well-differentiated MCTs were predominantly characterized by diffuse cytoplasmic (8/19) and membranous stain (7/19); a diffuse distribution of KIT positive cells was displayed in most of these tumors as well (15/19). Atypical MCTs showed diffuse distribution of labeled cells (4/7), and diffuse cytoplasm immunostaining was seen most (5/7). The pleomorphic MCT showed diffuse cytoplasmic KIT stain, with moderate labeling intensity, typically displaying focal distribution in deeper areas of the neoplasm. According to the results, there was no correlation between the type of MCTs and KIT expression, although the use of feline KIT immunohistochemistry could be useful to assess the mast cell origin.

Hypericum grandifolium Choisy: a species native to Macaronesian Region with antidepressant effect.

AIM OF THE STUDY: Various species of Hypericum genus have been used in the Canary Islands as sedative, diuretic, vermifuge, wound healing, antihysteric and antidepressant agent. Studies have shown that methanol extract of Hypericum grandifolium Choisy is active in tetrabenazine-induced ptosis and forced swimming tests. In the current study, the aqueous, butanol and chloroform fractions obtained from the methanol extract as well as three sub-fractions derived from the chloroform fraction were evaluated for their central nervous effects in mice, particularly their antidepressant activity. MATERIALS AND METHODS: The central nervous effect of different fractions and sub-fractions of Hypericum grandifolium was evaluated in mice using various behavioural models including locomotor and muscle relaxant activity, forced swimming test, effect on normal body temperature, barbiturate-induced sleep, tetrabenazine-induced syndrome and 5-hydroxytryptohan-induced head twitches and syndrome. RESULTS: We found that the butanol and chloroform fractions and all sub-fractions showed an antidepressant effect in the forced swimming test, the chloroform fraction being the most active. They produced no effects or only a slight depression of locomotor activity. Chloroform fraction significantly increased the pentobarbital-induced sleeping time, produced a slight but significant hypothermia and antagonized tetrabenazine-induced ptosis, whereas the butanol fraction produced a slight potentiation of 5-HTP-induced head twitches and syndrome. CONCLUSIONS: The present results, together with previous pharmacological and phytochemical data, indicated that Hypericum grandifolium possess antidepressant-like effects in mice and that different constituents, such as the flavonoids and the benzophenone derivatives, could be responsible at least in part for the antidepressant effects observed for this species.

Antidepressant activity of some Hypericum reflexum L. fil. extracts in the forced swimming test in mice.

We previously reported that oral administration of the methanol extract obtained from the aerial part in blossom of Hypericum reflexum L. fil. was active in the tetrabenazine and forced swimming test. In the present study, the effect of the aqueous, butanol and chloroform fractions obtained from the methanol extract of this species on the central nervous system was investigated in mice, particularly in animal models of depression. Antidepressant activity was detected in the butanol and chloroform fractions of this species using the forced swimming test since both fractions induced a significant reduction of the immobility time, producing no effects or only a slight depression on spontaneous motor activity when assessed in a photocell activity meter. Moreover, these fractions did not alter significantly the pentobarbital-induced sleeping time. On the other hand, the chloroform fraction produced a slight but significant hypothermia and was also effective in antagonizing the ptosis induced by tetrabenazine. Furthermore, the butanol fraction produced a slight potentiation of the head twitches and syndrome induced by 5-HTP. Taken together, these data indicate that the butanol and chloroform fractions from Hypericum reflexum possess antidepressant-like effects in mice, providing further support for the traditional use of these plants in the Canary Islands folk medicine against central nervous disorders.

Expression of androgen, oestrogen alpha and beta, and progesterone receptors in the canine prostate: differences between normal, inflamed, hyperplastic and neoplastic glands.

The expression of receptor for androgen (AR), oestrogen alpha and beta (ERalpha and ERbeta) and progesterone (PR) was examined immunohistochemically in canine prostate specimens (normal, hyperplastic, inflamed [prostatitis] or neoplastic). AR immunolabelling was seen in 100% of epithelial cells of normal and hyperplastic tissue, the corresponding figures for inflamed and carcinomatous tissue being 74% and 65%, respectively. ERalpha labelling was seen in 85% of epithelial cells in normal prostate glands, the corresponding figures for hyperplastic, inflamed and neoplastic glands being 35%, 22% and 12%, respectively. ERbeta labelling was seen in 85% of epithelial cells of normal glands and in about 70% of such cells in glands showing pathological changes. On the other hand, PR expression (weak) in normal glands was observed in fewer epithelial cells (44%) than in hyperplastic (70%), inflamed (62%) or neoplastic (64%) glands.

Evaluation of the analgesic and topical anti-inflammatory effects of Hypericum reflexum L. fil.

The present study investigates the analgesic and topical anti-inflammatory effects of the infusion, methanol extract and fractions of the aerial part in blossom of Hypericum reflexum L. fil. in mice. The acetic acid-induced writhing test, formalin test, tail flick test and the tetradecanoylphorbol acetate (TPA)-induced ear inflammation model in mice were used to determine these effects. Our findings show that oral administration of all extracts tested from this species significantly inhibit acetic acid-induced writhing in mice. Only the methanol extract and chloroform fraction were significantly active in both phases of formalin-induced pain and in the tail flick assays, suggesting that they may have central analgesic properties. On the other hand, the topical treatment of methanol extract, butanol and chloroform fractions of this species significantly reduced the TPA-induced ear oedema. In conclusion, the results indicate analgesic and topical anti-inflammatory activities in mice for the Hypericum species studied.

Antidepressant properties of some Hypericum canariense L. and Hypericum glandulosum Ait. extracts in the forced swimming test in mice.

It has been shown in a previous work that the methanol extract obtained from the aerial part in blossom of Hypericum canariense L. and Hypericum glandulosum Ait. was active in the tetrabenazine and forced swimming test. In the present study, the central nervous effect of the aqueous, butanol and chloroform fractions obtained from the methanol extracts of these Hypericum species was investigated in mice, particularly in animal models of depression. It was found that the immobility time in the forced swimming test was significantly reduced by the butanol and chloroform fraction of both species assayed, producing no effects or only a slight depression on spontaneous motor activity when assessed in a photocell activity meter. In this regard, the efficacy of the chloroform extract from Hypericum glandulosum Ait. (500 mg/kg p.o.) in the forced swimming test was comparable to that of the tricyclic antidepressant imipramine (50 mg/kg p.o.). In addition, the Hypericum glandulosum chloroform fraction was also effective in antagonizing the ptosis induced by tetrabenazine. Moreover, Hypericum canariense butanol fraction and Hypericum glandulosum chloroform fraction produced a slight but significant hypothermia. Taken together, these data demonstrate that the butanol and chloroform fractions from Hypericum canariense and Hypericum glandulosum possess antidepressant-like effects in mice, providing further support for the traditional use of these plants in the Canary Islands folk medicine against central nervous disorders.

Analgesic and topical anti-inflammatory activity of Hypericum canariense L. and Hypericum glandulosum Ait.

The present study investigates the analgesic and topical anti-inflammatory activities of the infusion, methanol extract and fractions of the aerial part in blossom of Hypericum canariense L. and Hypericum glandulosum Ait. in mice. The acetic acid-induced writhing test, tail flick test and the tetradecanoylphorbol acetate (TPA)-induced ear inflammation model in mice were used to determine these effects. Our findings show that oral administration of methanol extracts, and the aqueous, butanol and chloroform fractions of both species and the infusions of Hypericum glandulosum significantly inhibit acetic acid-induced writhing in mice. Only the infusion, methanol extract and butanol and chloroform fractions of Hypericum glandulosum were significantly active in the tail flick assay, suggesting that they may have central analgesic properties. On the other hand, the topical treatment of all extracts tested from both species, with the exception of the infusions and the Hypericum canariense aqueous fraction, significantly reduced the TPA-induced ear oedema. In conclusion, the results indicate analgesic and topical anti-inflammatory activities in mice for the Hypericum species studied.

Studies on the analgesic and anti-inflammatory effects of Sideritis candicans Ait. var. eriocephala Webb aerial part.

Different extracts and fractions from Sideritis candicans Ait. var. eriocephala Webb aerial part were investigated for their analgesic, anti-inflammatory and antimicrobial activities in mice. Results indicated that the extracts assayed showed anti-nociceptive activities because they were able to reduce the nociceptive response to chemical pain stimuli, such as in the acetic acid-induced writhing test. Moreover the extracts also possessed anti-inflammatory activity against carrageenan-induced paw oedema and TPA-induced ear oedema, being the chloroform fraction the most active. Further fractionation and analysis of this fraction revealed that the analgesic and anti-inflammatory activities found could be related in part to the presence of phytosterols, alpha and beta amyrin triterpenic derivatives and ent-kaurene type diterpenes in this species, since some of these compounds are endowed with these activities.

Differential expression of CD44 in canine melanocytic tumours.

CD44, the main cell surface receptor for hyaluronan (HA), is often overexpressed in tumour cells, and its presence has been related to cell proliferation and migration. Many of the functions of CD44 are mediated through its interaction with hyaluronan. This study investigated the expression of CD44 in CML-1 and CML-10c2 canine melanoma cell lines and melanoma biopsies, and the production of hyaluronan and versican by the canine melanoma cell lines. Versican is an extracellular proteoglycan that binds hyaluronan, forming a tridimensional pericellular coat surrounding the cells. Both canine melanoma cell lines expressed CD44 and produced HA, but only CML-1 produced versican. Cells expressing all three components (CD44, HA and versican) formed abundant extracellular matrices as demonstrated by a particle exclusion assay. CD44 was present within benign and malignant melanomas, but its expression was more intense in malignant melanomas (P < 0.01). In high CD44-expressing tumours, CD44 tended to be present in the periphery of malignant melanomas, whereas its expression was homogeneous in benign melanomas.

Rhabdomyosarcoma in a racing pigeon (Columba livia).

An adult racing pigeon (Columba livia) was presented with a subcutaneous mass on the ventral aspect of the right wing. A fine-needle aspirate and radiographic study of the mass were suggestive of highly invasive sarcomatous neoplasm. Euthanasia was decided because of the poor prognosis. Necropsy confirmed the highly invasive nature of the neoplasm, which also occupied a large portion of the right breast. There also was extensive osteolysis of the sternum with neoplastic invasion of the left breast and the coelomic cavity. Histopathology revealed a highly cellular, poorly demarcated, unencapsulated invasive sarcoma. Immunohistochemistry was positive for muscle actin, and myoglobin, weakly positive for vimentin, and negative for desmin, neuron-specific enolase and S-100 protein, suggesting a diagnosis of undifferentiated rhabdomyosarcoma.

Antimicrobial studies on three species of Hypericum from the Canary Islands.

The antimicrobial activity of several extracts and fractions of the aerial parts of Hypericum canariense, Hypericum glandulosum and Hypericum grandifolium was investigated using the disc diffusion and broth microdilution methods against twelve reference microorganisms (eight bacterial and four fungal strains). The methanol extract and chloroform fraction of H. canariense, as well as the methanol extracts, butanol and chloroform fractions of both H. glandulosum and H. grandifolium exhibited a good antibacterial activity against four Gram-positive bacteria (Bacillus cereus var. mycoides, Micrococcus luteus, Staphylococcus aureus and Staphylococcus epidermidis) and the Gram-negative bacterium Bordetella bronchiseptica with the diameters of growth inhibition area in the range 10-25 mm and MICs values between 0.03 and 0.29 mg/ml. Neither the infusions and aqueous fractions of the species studied nor the butanol fraction of H. canariense showed any antibacterial activity against the tested microorganisms. Amongst the active extracts, the minimum inhibitory concentration (MIC) determination showed that the H. canariense chloroform fraction was the most active against M. luteus, S. aureus and S. epidermidis. No antifungal activity was seen with any of the extracts or fractions tested. The results of this study support the use of these species in Canarian traditional medicine to treat skin infections.