Electron microscopic observations of stratum corneum intercellular lipids in normal and atopic dogs.

The barrier function of mammalian skin is maintained by intercellular stratum corneum lipids. In human patients with atopic dermatitis, an abnormal lipid barrier results in dry skin and increased transepidermal water loss. At this time, it is not known if a defective lipid barrier is present in atopic dogs. Normal and atopic canine skin were postfixed in ruthenium tetroxide and studied using transmission electron microscopy to determine structural differences within stratum corneum lipids. Intercellular lipid lamellae were graded on a semiquantitative scale. The deposition of stratum corneum lipid lamellae in atopic canine skin appeared markedly heterogeneous compared with that seen in normal canine skin. When present, the lamellae often exhibited an abnormal structure. The continuity and thickness of the intercellular lipid lamellae were significantly less in nonlesional atopic than in normal canine skin. These preliminary observations suggest that the epidermal lipid barrier is defective in atopic canine skin. Additional studies are needed to further characterize the biochemical defect and to possibly correct it with nutritional and/or pharmacologic intervention.     

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 14days 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.     

Is the skin barrier abnormal in dogs with atopic dermatitis?

In mammalian skin, the stratum corneum exerts a barrier function that protects from transepidermal water loss and the penetration of exogenous molecules, such as allergens, from the environment. Recently, skin barrier defects have been shown to be of prime importance in the pathogenesis of human atopic dermatitis. In this review, we summarize the latest research data pertinent to the stratum corneum and barrier function of dogs with atopic dermatitis. At the time of this writing, there is increasing evidence that a skin barrier defect likely exists in dogs with atopic dermatitis. This barrier dysfunction is characterized by abnormal intercellular stratum corneum lipid lamellae, abnormal stratum corneum morphology, reduced and abnormal ceramide content and, in some but not all dogs, abnormal filaggrin expression. In association with these changes, there is higher transepidermal water loss in atopic than in normal canine skin. Furthermore, atopic inflammation appears to worsen transepidermal water loss and filaggrin expression. It remains unknown, however, if the changes observed are primary (i.e. of genetic origin) or secondary to atopic inflammation that also exists even in clinically normal skin. Finally, whether or not a therapeutic intervention aimed at restoring a dysfunctional skin barrier is of any clinical benefit to atopic dogs has not yet been proven unequivocally.     

Phenotypic analysis for a cell line of canine epidermal keratinocytes

Epidermal keratinocytes have the potential to produce inflammatory mediators that are considered to play an important role in skin diseases such as atopic dermatitis (AD). Thus, cell lines of canine epidermal keratinocytes are useful for studying the biological reactivity of keratinocytes in vitro. However, there has been no report on properly analyzing the phenotype of canine keratinocyte cell lines. In this work, we performed phenotypic analysis of CPEK, which was derived from the epidermis of an adult dog in order to examine the phenotypic similarity with epidermal keratinocytes. The present findings indicated that CPEK cells expressed markers for epidermal keratinocytes including cytokeratin 14, alpha6 integrin and PCNA. Our findings demonstrated that CPEK could be a useful cell line for investigating the central role of epidermal keratinocytes in the pathogenesis of AD in vitro.     

Characterization of the canine skin barrier restoration following acute disruption by tape stripping

The stratum corneum (SC) forms the main part of the permeability barrier of the skin. In mice and in humans, cutaneous barrier disruption can be generated by removing the SC with tape stripping (TS) and the skin barrier function can be assessed by measurement of transepidermal water loss (TEWL). The aim of the present study was to characterize the skin barrier restoration in the dog following mechanical disruption and to analyse the correlation between the skin barrier recovery and TEWL measurement. Thirty sequential TS were performed on 12 sites on four healthy beagle dogs. The number of TS was chosen to ensure a sufficient barrier disruption with a slow recovery. Skin repair was assessed for 72 h by clinical and histological examinations, and TEWL measurements. The results showed that performing 30 TS was adequate to disrupt the skin barrier in the dog. The homeostatic repair response, initiated in the skin, was rapid and characterized by complete restoration of the SC within 72 h, accompanied by greater basal cell proliferation, and dermal eosinophilic inflammation. TEWL was significantly increased by complete removal of the SC but recovered along with restoration of the SC (Scheffe test, P ≤ 0.05). Characterization of a canine model of barrier disruption and restoration and assessment of the skin barrier function by TEWL measurements could help better understand the events implied in skin barrier function. Development of this canine model is also necessary for future studies on the effects of treatments aimed at restoring the skin barrier.     

The Structure and Function of Epidermal Lipids

Abstract— This review article examines current knowledge of the structure and function of epidermal lipids in human and veterinary dermatology. Dynamic transformations in lipid composition and structure occur as cells migrate through the epidermis. The lipids which are formed have a direct effect on epidermal functions. Ceramides appear to be the most important lipid component for lamellar arrangement in the stratum corneum and barrier protection. Polyunsaturated fatty acids in the epidermis are also important because they are incorporated into the ceramides. Additionally, arachidonic acid is bound to the phospholipid portion of cell membranes and is the most important precursor of endogenous eicosanoids. The eicosanoids are vital for epidermal homeostasis and play an important role in the pathogenesis of inflammatory dermatoses when produced in abnormal quantities. In addition to barrier protection, other vital epidermal functions associated with the lipids include stratum corneum water-holding, cohesion and desquamation of corneocytes, and control of epidermal proliferation and differentiation.     

Characterization and quantification of ceramides in the nonlesional skin of canine patients with atopic dermatitis compared with controls

As in humans, there is mounting evidence in support of an abnormal skin barrier contributing to the pathogenesis of canine atopic dermatitis (AD). Studies in people with AD have associated an abnormal skin barrier with deficiencies in ceramides, which represent important components of the stratum corneum (SC) intercellular lipid lamellae. Therefore, the goal of this study was to determine if the SC of dogs with AD is deficient in ceramides compared to normal dogs. Samples of SC were obtained from nonlesional skin of the caudal abdomen of 14 patients with AD and 14 age-, breed- and sex-matched healthy controls using a cyanoacrylate stripping procedure, and the subclass and relative amount of ceramides were assessed blindly by thin layer chromatography. Paired t -tests using R statistical computer software revealed the percentage amounts of ceramides 1 and 9 were significantly lower in nonlesional skin of AD dogs compared to controls ( P = 0.034 and P = 0.047, respectively), and the cholesterol percentage amount was significantly higher in AD dogs than in controls ( P = 0.016). Furthermore, the cholesterol/ceramide ratio was significantly higher in the AD group with respect to controls ( P = 0.014). These findings suggest that decreased amounts of ceramides in the skin of dogs with AD may be involved in the impaired barrier function of their skin.     

Micromorphology of the skin (epidermis, dermis, subcutis) of the dog

Grossly, the skin of the dog is characterized by varying thicknesses, density of the hair coat and the implantation angle of the hairs. Multiple folds varying in shape are formed by the epidermis and the papillary layer of the corium. The epidermis consists of a stratum profundum with a continuous layer of basal cells, a stratum spinosum consisting of 3-6 cell layers, a stratum superficiale with single granulosa and "lucidum" cells and a well-developed stratum corneum. The corium shows a narrow stratum papillare corli containing delicate fibres and a broad stratum reticulare corii with mainly coarse fibre bundles. The subcutis is divided into adipose and fibrous layers. The broad adipose layer displays breed and individual differences which vary with the region of the body. The fibrous layer corresponds to the connective tissue layer, formerly described as fascia trunci superficialis. The morphological and functional unity of the corium and subcutis is discussed.     

Characterization of canine filaggrin: gene structure and protein expression in dog skin

Background – Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. Hypothesis/Objectives – The aim of this study was to determine the structure of the canine FLG gene and to raise anti‐dog FLG antibodies, which will be useful to detect FLG protein in dog skin. Methods – The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti‐dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. Results – Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti‐dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. Conclusions and clinical importance – This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti‐dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG‐deficient dogs with canine atopic dermatitis or ichthyosis.     

Transepidermal water loss (TEWL) reflects skin barrier function of dog

The correlation between skin barrier function and transepidermal water loss (TEWL) was evaluated in dogs. Stratum corneum (SC) of 10 healthy dogs was removed by tape stripping (TS), which decreased the corneal layer to allow for permeation of fluorescent dye into skin. TEWL of damaged skin was measured with the closed-chamber-type TEWL analyzer, CC-01. The frequency of TS was directly related to the decrease of SC and the increased permeation of fluorescent dye, and TEWL increased with increasing impairment of skin barrier function. The results suggest that increased TEWL reflects impaired canine skin barrier function.     

The role of allergy in the development of canine pyoderma

Pyoderma is commonly seen in canine veterinary practice and usually occurs as a complicating factor in other primary conditions. Allergic skin disease is often the underlying cause but the precise relationship between allergy and infection is unknown. Our studies have investigated the relationship between bacterial proliferation at the skin surface and hypersensitivity reactions with the skin. Hypersensitive dogs were shown to have significantly higher surface counts of staphylococci than normal controls and these bacteria were concentrated in the more superficial layers of the stratum corneum. Intradermal injection of staphlococcal antigens in normal dogs elicited epidermal damage similar to that seen in clinical disease. Preliminary autoradiographic studies using a model of canine skin hypersensitivity reactions showed that percutaneous absorption of radiolabelled staphylococcal antigens was increased by mast cell degranulation. These findings suggest that a major role of hypersensitivity reactions in the pathogenesis of pyoderma may be via an effect on epidermal permeability, promoting penetration of staphylococcal antigens from the stratum corneum which then cause the lesions of pyoderma. Hypersensitivity reactions may also lead to changes in the skin surface microclimate leading to increased bacterial counts on the skin surface, so exacerbating the condition.     

Structure of the epidermis in Ayrshire bullocks.

The living epidermis and stratum corneum of cattle, when measured on cryostat sections, were both found to be approximately 30 micrometer thick. However, the corneum contained about 30 cell layers, whereas the living epidermis was composed of only four. Histological processing involving fixation, dehydration and paraffin embedding, resulted in the loss of half of the stratum corneum and proved to be unsuitable for study of this layer. Lipid derived mainly from the sebaceous gland was present between the squames in the outer two thirds of the corneum probably in the form of an emulsion with sweat.     

Experimental bovine Trichophyton verrucosum infection. Comparison of the rate of epidermal cell proliferation and keratinisation in non-infected and reinoculated cattle.

The rate of epidermal cell renewal in normal bovine skin and that reinoculated with Trichophyton verrucosum was measured using a radioautographic technique. The transit times of both nucleated and fully keratinised cells were measured in sequential biopsy samples removed at predetermined periods after intradermal inoculation with radio-labelled isotopes. The total time taken for cells of the basal layer to travel to the point of desquamation in the stratum corneum was 18 days in normal cattle. In similar areas on cattle that had been reinoculated with T verrucosum the total epidermal cell renewal time was reduced to 12 days. Increased protein synthesis, as measured by incorporation of radio-labelled nucleoside was evident in basal cells within 24 h of reinoculation with the fungus. The nucleated epidermal cell thickness had almost doubled in areas of reinoculated skin within 72 h and increased cell proliferation was maintained for at least 10 days. Desquamation of the thickened stratum corneum had occurred within seven days of reinoculation with the fungus.     

Transmission electron microscopy studies in an experimental model of canine atopic dermatitis

Impairment of skin barrier function has been hypothesized in canine atopic dermatitis (AD). In this prospective, controlled study, the ultrastructure of the upper epidermal layers was investigated using an experimental model of canine AD. Seven atopic Beagles sensitized to Dermatophagoides farinae and four healthy Beagles were used as controls. Both normal and atopic dogs were challenged with D. farinae for 3 days. Clinical signs were scored and skin biopsies were taken from the inguinal area before and 3 days after allergen exposure. Samples were processed to enhance lipid visibility and evaluated by Transmission Electron Microscopy. Emphasis was placed on evaluation of the lipid lamellae (LL), and lamellar bodies (LB) of the stratum corneum.
After allergen challenge, atopic Beagles developed severe pruritic dermatitis while no skin lesions were noted in the controls. Ultrastructurally, before allergen challenge, atopic Beagles displayed focally severe abnormalities in LL organization and wider intercellular spaces containing abnormal lipid material. In atopic Beagles, LBs were frequently found inside corneocytes while this finding was not observed in the controls. After allergen challenge, further increase of intercellular spaces was observed in the stratum corneum of atopic Beagles while no appreciable changes were observed in the normal dogs. Intercellular spaces in atopic Beagles were filled with abundant amounts of abnormal lipid material and highly disorganized LL. It is concluded that baseline differences in the ultrastructure of the skin exist between normal and experimentally sensitized atopic Beagles and that these changes are aggravated by allergen challenge and the resulting flare-up of dermatitis.     

Measurement of skin hydration in normal dogs and in dogs with atopy or a scaling dermatosis

The role of the stratum corneum both as a barrier to water loss from the body and as a biological microenvironment is well recognised, although most discussion of these topics is expressed in qualitative terms. This paper describes the use of techniques for the measurement of some aspects of the skin microclimate, and their application to canine skin. The techniques used were the assessment of skin moisture, hygroscopicity and water-holding capacity by measurement of skin electrical capacitance. Meaningful deductions regarding the water dynamics of certain skin sites can be drawn, and it was shown that significant differences in hydration exist between different skin sites. The techniques were also used to show that scaly skin is ‘drier’ than normal skin, and that the water dynamics of the non-lesional skin of atopic dogs does not differ from that of normal dogs.     

盐胁迫下2种樱桃叶片解剖结构的变化

为了筛选耐盐樱桃砧木,用于天津市周边轻度盐碱地区栽培,本试验通过徒手切片的方法,研究了两种樱桃砧木在盐胁迫下的叶片解剖结构变化。结果表明：在盐胁迫下,四川樱桃的叶片增厚了3.99%,大青叶的减少了2.68%;大青叶的表皮层细胞增厚了23.6%,四川樱桃的减少了3.84%;大青叶的角质层增厚3.57%,四川樱桃的减少了18.31%;四川樱桃的栅栏组织层变化不大,大青叶的减少了16.42%;四川樱桃的海绵组织层增厚了31.96%,大青叶的增厚了21.27%;四川樱桃的栅栏组织和海绵组织层厚度的比值变小、减少了29.17%,大青叶的减少了20.80%。盐胁迫使大青叶叶片表皮细胞层变厚,角质层加厚,栅栏组织层变薄,海绵组织层加厚,整个叶片变薄。盐胁迫使四川樱桃表皮细胞层变薄,角质层变薄,海绵组织层加厚,整个叶片变厚。从盐胁迫下的2种樱桃叶片结构的变化综合分析认为大青叶的耐盐性明显高于四川樱桃。     

Ichthyosis and hereditary cornification disorders in dogs

The stratum corneum (SC), the outermost layer of the epidermis, serves a crucial role in maintaining body hydration and protection from environmental insults. When the stratum corneum is injured or when the genetic blueprints are flawed, the body is at risk of dehydration, secondary infections and allergen sensitization. Advancements in veterinary dermatology have revealed a wide gamut of disease from relatively benign to lethal that specifically arise from flawed structural proteins, enzymes or lipids needed to create the corneocytes and lipid bilayers of the SC. Some conditions closely mimic their human counterparts while others are unique to the dog. This review will focus on forms of ichthyosis in the dog.     

Role of Cumulus Cells on In Vitro Maturation of Canine Oocytes

The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.