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.

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.

Immunohistochemical study of doublecortin and nucleostemin in canine brain.

Finding a marker of neural stem cells remains a medical research priority. It was reported that the proteins doublecortin and nucleostemin were related with stem/progenitor cells in central nervous system. The aim of the present immunohistochemical study was to evaluate the expression of these proteins and their pattern of distribution in canine brain, including age-related changes, and in non-nervous tissues. We found that doublecortin had a more specific expression pattern, related with neurogenesis and neuronal migration, while nucleostemin was expressed in most cells of almost every tissue studied. The immunolabeling of both proteins decreased with age. We may conclude that nucleostemin is not a specific marker of stem/progenitor cells in the dog. Doublecortin, however, is not an exclusive marker of neural stem cells, but also of neuronal precursors.

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.

Effects of Essential Oils and Polyunsaturated Fatty Acids on Canine Skin Equivalents: Skin Lipid Assessment and Morphological Evaluation.

A canine skin equivalent model has been validated for the assessment of a topical formulation effects. Skin equivalents were developed from freshly isolated cutaneous canine fibroblasts and keratinocytes, after enzymatic digestion of skin samples (n = 8) from different breeds. Fibroblasts were embedded into a collagen type I matrix, and keratinocytes were seeded onto its surface at air-liquid interface. Skin equivalents were supplemented with essential oils and polyunsaturated fatty acid formulation or with vehicle. Skin equivalents were histopathologically and ultrastructurally studied, and the three main lipid groups (free fatty acids, cholesterol, and ceramides) were analyzed. Results showed that the culture method developed resulted in significant improvements in cell retrieval and confluence. Treated samples presented a thicker epidermis with increased number of viable cell layers, a denser and compact stratum corneum, and a more continuous basal membrane. Regarding lipid profile, treated skin equivalents showed a significant increase in ceramide content (51.7 ± 1.3) when compared to untreated (41.6 ± 1.4) samples. Ultrastructural study evidenced a compact and well-organized stratum corneum in both treated and control skin equivalents. In conclusion, cell viability and ceramides increase, after lipid supplementation, are especially relevant for the treatment of skin barrier disruptions occurring in canine atopic dermatitis.

In vitro development and characterization of canine epidermis on a porcine acellular dermal matrix.

The aim of this study was to develop and to characterize a canine skin epidermal model able to form a proper epidermis on a porcine acellular dermal matrix (PADM). In addition, the role of fibroblasts in skin barrier formation was studied by incorporating or omitting canine dermal fibroblasts in the PADM. Canine epidermal composites were developed by seeding keratinocytes onto the surface of PADM that were previously seeded or non-seeded with dermal fibroblasts. After 14 days of culture under air-exposed conditions and in a special growth medium, skin composites were histologically processed and immunohistochemically characterized to determine the expression of cytokeratins and of vimentin and the presence of basement membrane. In all composites, keratinocytes underwent differentiation to a multilayer epidermis with 5-7 viable cell layers. The stratum basalis, stratum spinosum, stratum granulosum and stratum corneum were identified. The expression of cytokeratins was similar to that described in healthy canine epidermis. Laminin and collagen IV immunostaining revealed a homogeneous layer in the epidermal-dermal junction only when the matrix had been seeded by canine dermal fibroblasts. The model may become a simple, useful and cost-effective tool to investigate the biology and pathology of canine epidermis and could partially replace animal testing in several areas of dermatological research.

Immunohistochemical detection of COX-2 in feline and canine actinic keratoses and cutaneous squamous cell carcinoma.

Cyclooxygenase-2 (COX-2) overexpression and its causal role in epidermal carcinogenesis have been demonstrated in human actinic keratoses (AK) and cutaneous squamous cell carcinoma (SCC). The aim of this study was to determine immunohistochemically the level of expression of COX-2 in feline and canine AK (n=18), SCC (n=36) and inflammatory dermatoses (n=24). COX-2 immunoreactivity was detected in all feline and canine SCC. In all specimens, labelled basal and suprabasal neoplastic keratinocytes were localized within and below areas of superficial erosion or ulceration and only scattered deeper tumour cells were positively labelled. In most cases, positive immunoreactivity of keratinocytes was associated with the presence of granulocytes. COX-2 expression was detected in 3/9 canine and 4/9 feline cases of AK and in only one case was associated with inflammation. Inflammatory dermatoses were characterized by positively labelled epidermal and follicular basal and suprabasal keratinocytes that were always associated with granulocyte exocytosis. These results indicate that further study of the effect of using COX-2 inhibitors in the management and prevention of feline and canine cutaneous SCC is warranted. The association between inflammatory cells and COX-2 expressing epidermal cells opens a new line of research regarding the role of COX-2 in SCC oncogenesis. Moreover, further studies should investigate the role of COX-2 in the pathogenesis and management of AK in animals.

Acral mutilation syndrome in a miniature pinscher.

Acral mutilation syndrome (AMS) is a rare canine hereditary sensory neuropathy that results in progressive mutilation of the distal extremities and which has been reported only in German short-haired pointers, English pointers, English springer spaniels and French spaniels. The present report describes a case of AMS in an 18-month-old female miniature pinscher with progressive self-mutilation of the hind feet. The dog did not respond to any treatment and was humanely destroyed at the age of 30 months. Microscopical findings post mortem were restricted to the nervous system and were compatible with AMS. This is the first case of AMS described in a miniature pinscher. It is not known if the disease was the result of a point mutation in this particular dog or if the miniature pinscher breed will evolve to become a breed predisposed to AMS.

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.

Cutaneous mucinosis in shar-pei dogs is due to hyaluronic acid deposition and is associated with high levels of hyaluronic acid in serum.

Cutaneous mucinosis affects primarily shar-pei dogs. Hyaluronic acid (HA) is considered to be the main component of mucin and CD44 is the major cell surface receptor of HA, necessary for its uptake and catabolism. The aims of this study were to identify the composition of the mucin in cutaneous mucinosis of shar-pei dogs, investigate the correlation between the deposition of HA and the expression of CD44, and determine whether shar-pei dogs with cutaneous mucinosis presented with elevated levels of serum HA. In skin biopsies, the mucinous material was stained intensely with Alcian blue and bound strongly by the hyaluronan-binding protein. No correlation was found between the degree of HA deposition in the dermis and the expression of CD44 in the skin of shar-pei dogs affected or unaffected by cutaneous mucinosis. A clear positive correlation was found between the existence of clinical mucinosis and the serum HA concentration. In control dogs, serum HA ranged from 155.53 to 301.62 microg L(-1) in shar-pei dogs; without mucinosis it ranged from 106.72 to 1251.76 microg L(-1) and in shar-pei dogs with severe mucinosis it ranged between 843.51 to 2330.03 microg L(-1). Altogether, the results reported here suggest that mucinosis of shar-pei dogs is probably the consequence of a genetic defect in the metabolism of HA.

Canine cutaneous spindle cell tumours with features of peripheral nerve sheath tumours: a histopathological and immunohistochemical study.

In veterinary medicine, the term peripheral nerve sheath tumour is usually restricted to neoplasms that are closely associated with an identified nerve. Thirty-three cases of canine cutaneous tumours previously classified as spindle cell tumours with features resembling peripheral nerve sheath tumours were examined. Two histological patterns were identified: dense areas of spindle shaped cells resembling the Antoni A pattern and less cellular areas with more pleomorphic cells resembling the Antoni B pattern. Immunohistochemically, all tumours uniformly expressed vimentin and 15/33 (45.4%) had scattered and patchy expression of S-100. Laminin expression was found in 25/33 (75.7%) tumours and collagen IV labelling occurred in 14/33 (42.4%). Expression of protein gene product 9.5 was detected in 31/33 (93.9%) of tumours and neuron specific enolase labelling was present in 27/33 (81.8%). Glial fibrillary acidic protein was only expressed within the cytoplasm of some large multinucleated cells in one tumour. These findings suggest that any cutaneous tumour with one of the two histopathological patterns described above should be described as a cutaneous peripheral nerve sheath tumour and that expression of S-100, laminin and collagen IV may be used to define a schwannoma.

Histopathological features of ocular leishmaniosis in the dog.

Canine leishmaniosis (CL) can present with multiple clinical signs and ocular disease is reported to occur in almost 25% of affected dogs. The purpose of the present study was to characterize the nature of inflammation within the eyes of dogs with leishmaniosis and to determine whether parasites were present in these lesions. Eyes from 60 dogs with confirmed leishmaniosis that died or were humanely destroyed over a 4 year period were included in the study. Sections of formalin-fixed globes were stained with haematoxylin and eosin (HE) and subjected to immunohistochemistry using a Leishmania-specific antibody. Clinically evident ocular signs were present in 15 of 60 dogs (13 bilaterally and 2 unilaterally). Thirty-five of 60 dogs received some form of anti-protozoal treatment. In 36 of 120 eyes (30%) a granulomatous inflammatory infiltrate was found and in 32 of 120 eyes (26.6%) the parasite was identified immunohistochemically within the globe. Ocular tissues affected, in order of frequency, were conjunctiva and limbus, ciliary body, iris, cornea, sclera and iridocorneal angle, choroid and the optic nerve sheath. Different microscopical patterns were defined in each of these structures. Leishmania organisms and associated inflammation can be found in different ocular tissues, accounting for some of the ocular clinical signs described for this disease.

Evaluation of the expression of P-selectin, ICAM-1, and TNF-alpha in bacteria-free lesional skin of atopic dogs with low-to-mild inflammation.

Canine atopic dermatitis (AD) is a pruritic skin condition that shares many clinical and pathophysiological features with its human counterpart. A major therapeutic challenge of AD is the control of the skin inflammatory process. A detailed knowledge of the pro-inflammatory molecules involved in cell recruitment in AD would allow for a better control of the disease. We thus have studied the protein expression of P-selectin, ICAM-1 and TNF-alpha in the lesional and non-lesional skin of atopic dogs that had been treated for bacterial infections. Despite a low-to-mild inflammatory process, P-selectin protein was clearly upregulated in the lesional skin areas when compared with non-lesional skin (four-fold average increase). This P-selectin upregulation was accompanied by signs of functional changes such as increased cell margination, and membrane-associated protein expression. Although the expression of ICAM-1 and TNF-alpha was not enhanced in the lesional versus the non-lesional skin, there was a trend towards a correlated upregulation of both molecules. Further studies will help elucidate the significance of the substantial overexpression of P-selectin in canine AD, in particular in a scenario where bacterial antigens are not contributing as pro-inflammatory stimuli.

PCR technique detection of Leishmania spp. but not Mycobacterium spp. in canine cutaneous 'sterile' pyogranuloma/granuloma syndrome.

Cutaneous 'sterile' pyogranuloma/granuloma syndrome (SPGS) is an uncommon canine skin disorder of unknown aetiopathogenesis. Histopathological findings and failure to demonstrate an aetiologic agent are suggestive of this syndrome. Nevertheless, it has been hypothesized that SPGS may be related to an immune response against persistent endogenous or exogenous antigens. The presence of Leishmania and Mycobacterium organisms was investigated by polymerase chain reaction (PCR) techniques in 46 canine skin samples histopathologically diagnosed as SPGS. Concomitantly, an immunohistochemical technique for Leishmania detection was applied on the same samples and the results were compared with those from PCR. The PCR technique yielded positive results for Leishmania spp. in 21 out of 46 skin samples. The results of immunohistochemical techniques were identical to those obtained by PCR. The PCR technique gave negative results for Mycobacterium spp. in all the samples examined. These results suggest the importance of looking for Leishmania spp. in skin biopsies with histopathological findings consistent with the diagnosis of SPGS.

Histological and immunohistochemical study of clinically normal skin of Leishmania infantum-infected dogs.

Skin lesions are the most usual manifestation of canine leishmaniosis. The aim of this study was to investigate the histological pattern and parasite load in clinically normal skin of Leishmania-infected dogs. Two groups of Leishmania-infected dogs were studied. Group A consisted of 15 symptomless animals which, although seronegative or only mildly seropositive, gave a positive polymerase chain reaction (PCR) for Leishmania in the skin. Group B consisted of 20 clinically affected dogs which were highly seropositive and PCR-positive. Biopsies of normal skin from all dogs were processed for routine histology and Leishmania immunohistochemistry. The study demonstrated microscopical lesions and the presence of parasites in the skin from dogs of group B, but not group A. The results cast doubt on the relevance of infected but symptomless dogs in the epidemiology of canine leishmaniosis. In contrast, however, the clinically normal skin of sick dogs harbours the parasite and probably plays a role in the transmission of leishmaniosis.

Piecemeal degranulation (PMD) morphology in feline circulating eosinophils.

Buffy coat preparation from six cats with 600-4560 circulating eosinophils/microL was collected by either blood centrifugation or sedimentation, fixed in 2.5% glutaraldehyde, post-fixed in either 1% osmium or in 1.5% potassium ferrocyanide-reduced osmium, ultra-sectioned and examined by transmission electron microscopy. Ultrastructural changes of piecemeal degranulation (PMD), which is a mechanism of eosinophil granule contents release indicative of eosinophil activation, were observed in specific granules from all the samples examined. The spectrum of PMD included coarsening of the granule matrix, budding vesicles, fragmented granule cores and lucent granules. The number of presumably activated eosinophils with ultrastructural evidence of PMD did not correlate with the level of eosinophilia. The lack of correlation suggested that, analogously with humans, blood eosinophil count might not represent the best criterion to evaluate the contribution of eosinophils to tissue damage in certain feline eosinophil-associated diseases.

Assessment of proliferative activity of canine dermal mast cells by bromodeoxyuridine and proliferating cell nuclear antigen labelling.

Cutaneous mast cells from skin biopsies of three healthy dogs and three dogs with atopic dermatitis were assessed for their proliferative potential using bromodeoxyuridine and proliferating cell nuclear antigen labelling. Mast cells isolated from the skin of two healthy dogs were also studied using bromodeoxyuridine labelling. Mast cells in skin biopsy specimens and mast cells isolated from the skin of healthy dogs did not incorporate bromodeoxyuridine. Two mast cells expressing proliferating cell nuclear antigen were seen around two superficial vessels in the dermis of one atopic dog. Epidermal cells, glandular epithelial cells, fibroblasts and endothelial cells incorporated bromodeoxyuridine and showed positive staining for proliferating cell nuclear antigen. These results suggest that canine mature mast cells do not proliferate in the dermis.