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.     

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.     

Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof

Reasons for performing study: Abnormal epidermal stem cell regulation may contribute to the pathogenesis of equine chronic laminitis. Objective: To analyse the involvement of p63, a regulator of epidermal stem cell proliferative potential, in chronic laminitis. Methods: Epidermal tissues from skin, coronet and lamellae of the dorsal foot were harvested from 5 horses with chronic laminitis and 5 control horses. Tissues were analysed using histopathology, immunofluorescence microscopy and quantitative immunoblotting Results: Hoof lamellae of laminitic horses had a lower frequency of p63 positive cells than control lamellae, particularly in the distal region. Quantitative immunoblotting confirmed reduced p63 expression in the laminitic distal lamellar region. The decreased p63 expression in laminitic epidermal lamellae was most apparent in the abaxial region adjacent to the hoof wall and highly associated with the formation of terminally differentiated, dysplastic and hyperkeratotic epidermis in this region, whereas lamellae from control horses maintained high p63 expression throughout the axial‐abaxial axis. Conclusions: Expression of p63 in equine skin resembles that reported in other species, including man and rodents, suggesting that p63 can serve as a marker for the proliferative potential of equine epidermal stem cells. p63 expression was significantly lower in the chronic laminitic hoof than in that of control horses, suggesting laminitic hoof epithelium has more limited proliferative potential with a shift towards differentiation. This may reflect reduced activity of epidermal stem cells in laminitic hoof. It is proposed that p63 contributes to the maintenance of hoof lamellae and that misregulation of p63 expression may lead to epidermal dysplasia during lamellar wedge formation. Potential relevance: This study suggests that loss of epidermal stem cells contributes to the pathogenesis of equine laminitis. Autologous transplantation of p63‐positive epidermal stem cells from unaffected regions may have regenerative therapeutic potential for laminitic horses.     

Glucose transport in the equine hoof

Reasons for performing study: Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin‐responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. Objectives: To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. Methods: Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2‐deoxy‐D‐[2,6‐³H]glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin‐induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushing's syndrome. Results: Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin‐independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. Conclusions: The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1‐mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. Potential relevance: Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.     

Ultrastructure and histochemical study of glycoconjugates in the gills of the white croaker (Micropogonias furnieri)

The ultrastructure of the primary and secondary lamellae of gills was investigated in a marine teleost, the white croaker. The following cells were identified and briefly described: pavement cells, mucous cells, mitochondria-rich cells and rodlet cells. These cell types are present throughout the length of the lamellae. They are studied by means of a series of carbohydrate histochemical methods, including lectin procedures. Neutral sugars and substituted sialic acid were detected by means of periodic acid-borohydride reduction-saponification-periodic acid Schiff reaction (PA/Bh/KOH/PAS), saponification-selective periodic acid Schiff reaction (KOH/PA*S) and saponification-selective periodic acid-borohydride reduction-periodic acid Schiff reaction (KOH/PA*/Bh/PAS) histochemical techniques. A battery of seven lectins was used to study binding on tissue sections at the light microscopic level to characterize glycoconjugates in gills. The reaction to Canavalia ensiformis agglutinin (Con-A), Triticum vulgaris agglutinin (WGA), and Ricinus cummunis agglutinin-1 (RCA-1) was weak in pavement cells; unlike Con-A, the reaction to WGA and RCA-1 was more intense in mucous cells. Arachis hypogaea agglutinin (PNA) lectin showed a strong reaction in mucous cells. Ulex europaens agglutinin-1 (UEA-1) lectin was negative in all cell types. The lectin pattern was similar for both primary and secondary lamellae, except for PNA reaction, which was weak in the pavement cells of the secondary lamella and negative in the pavement cells of the primary lamella.     

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.     

Ultrastructural characterization of gills in juveniles of the Argentinian Silverside, Odontesthes bonariensis (Valenciennes, 1835) (Teleostei: Atheriniformes)

An ultrastructural study was performed on the gills of juvenile Argentinian silverside, Odontesthes bonariensis. The gills are composed of two sets of four holobranchs and, in turn, each holobranch consists of a gill arch and two rows of caudolaterally projecting branchial filaments. From the dorsal and ventral surfaces of each filament, branchial lamellae radiate out as foldings of the epithelial layer. Gill rakers are present on each of the gill arches, on the anteromedial side of the arch opposite to the filaments. Gill rakers, gill arches and branchial filaments are covered by a stratified epithelium, whereas branchial lamellae essentially consist of a thin epithelial envelope containing capillaries. In the stratified epithelium, mucous cells, rodlet cells, granular cells, pavement epithelial cells and mitochondria-rich cells are identified. The thin epithelium that lines the lamellae comprises two cell types, outer and inner epithelial cells, and the capillary walls on the inside of the epithelial envelope are defined by pillar cells. The ultrastructure of all these cell types is described and our findings are discussed in light of the existing data on fish gill morphology. In the gills of juvenile Argentinian silverside is of particular interest the characteristics showed by mitochondria-rich cells, such as their arrangement in clusters of 2-3 cells and their small and depressed surface in contact with the aquatic milieu, features which strongly resemble those of euryhaline species.     

The outer membrane of the avian egg shell as a reticular structure

The shell membranes of fowl, duck, quail and turkey eggs were examined by electron microscopy. A proportion of the outer membrane fibres from all sources were interconnected by lamellae about 90 A thick. The lamellae, which are probably protein, endow the outer membrane with a secondary reticulum reinforcing the reticulum formed by the fibres themselves.     

A scanning electron microscopic study of the vascular system of the bovine hind limb claw

The vascular organisation of the hind limb claws of clinically normal cattle was studied using scanning electron micrographs of plastic corrosion casts. The microvasculature of the dermal papillae and lamellae was demonstrated. A consistent vascular pattern was observed in the dermal papillae of the periople, coronary margin, sole and bulb. Ultrastructurally, the papillar vasculature consisted of a central artery and vein surrounded by a fine network of interconnected capillaries and venules. Arteriovenous anastomoses, connecting the central artery and vein, and peripherally situated arteriovenous loops were recognised in the terminal papillae of the white zone only. Arteriovenous anastomoses, focal enlargements and distension of the capillary bed were located within the interior half of the dermal lamellae. Numerous arteriovenous anastomoses were present throughout the entire dermis of the claw and were situated predominantly at the base of the dermal papillae and lamellae. The anastomoses and focal capillary enlargements may influence the blood flow in normal conditions and may play a significant role in the pathophysiology of bovine laminitis and sole ulceration.     

The ultrastructure of the subcommissural organ ependyma of the goat

The cells of pseudostratified columnar ciliated ependyma of the subcommissural organ in the goat were classified into two types on the basis of the distribution of chromatin material and nuclear clefts. Amongst the cell organelles the endoplasmic reticulum was highly developed and formed three types of Nebenkerne systems. The type-I Nebenkerne had spiral concentric lamellae and was associated with round lipid droplets. The type-II Nebenkerne, with widely spaced coils, was expanded towards its central and peripheral parts. The type-III Nebenkerne, composed of a meshwork of lamellae, was modified into a vacuolated form. The concentration of mitochondria was greatly increased towards the basal processes of the ependymal cells. The inclusion bodies included small electron-dense bodies, osmiophilic asteroid droplets, large round to spherical bodies and large round osmiophilic bodies with inner structures.     

A preliminary study into the correlation of stiffness of the laminar junction of the equine hoof with the length density of its secondary lamellae

Reason for performing study: The relationship between mechanical behaviour and microscopic structure of the laminar junction of equine hooves under testing conditions requires elucidation. Objectives: To determine mechanical parameters and 2D length density of profiles of secondary lamellae of the laminar junction in the dermal region and to assess possible correlations. Methods: Specimens (25 samples in total) of the laminar junction were taken from front, quarter and heel parts from 3 equine hooves and exposed to a uniaxial tensile test until rupture to obtain Young's moduli of elasticity, ultimate stress and strain. Neighbouring specimens to those used for the biomechanical experiment were processed histologically to assess the length density of laminar junction basement membrane using stereological grids. Results: The estimated median (interquartile range) length density of the laminar junction basement membrane was 0.024 (0.020–0.027)/µm. Young's modulus of elasticity was 0.15 (0.11–0.35) MPa in the small deformation region, and 7.58 (6.14–8.68) MPa in the linear region was. The ultimate stress was 1.67 (1.41–2.67) MPa, and the ultimate strain was 0.50 (0.38–0.70). The Young's modulus of elasticity in the region of small deformations has a moderate correlation with the length density of the laminar junction basement membrane. Conclusions: As with most soft biological tissues, the laminar junction has a nonlinear mechanical behaviour. Within the range of small deformations, which correspond to physiological loading of the laminar junction, a higher length density of the laminar junction basement membrane is correlated with a higher resistance of the laminar junction against high stresses transmitted from the distal phalanx to the hoof wall. Potential relevance: The condition of the laminar junction apparatus may be easily quantified as the length density of profiles of secondary dermal lamellae. This quantification provides a simple tool that could be used for comparing the proneness of the various parts of the laminar junction to initial stages of laminitis.     

The effect of hoof angle variations on dorsal lamellar load in the equine hoof

Reasons for performing study: In the treatment of laminitis it is believed that reducing tension in the deep digital flexor tendon by raising the palmar angle of the hoof can reduce the load on the dorsal lamellae, allowing them to heal or prevent further damage. Objective: To determine the effect of alterations in hoof angle on the load in the dorsal laminar junction. Methods: Biomechanical finite element models of equine hooves were created with palmar angles of the distal phalanx varying from 0–15°. Tissue material relations accounting for anisotropy and the effect of moisture were used. Loading conditions simulating the stages in the stance where the vertical ground reaction force, midstance joint moment and breakover joint moment were maximal, were applied to the models. The loads were adjusted to account for the reduction in joint moment caused by increasing the palmar angle. Models were compared using the stored elastic energy, an indication of load, which was sampled in the dorsal laminar junction. Results: For all loading cases, increasing the palmar angle increased the stored elastic energy in the dorsal laminar junction. The stored elastic energy near the proximal laminar junction border for a palmar angle of 15° was between 1.3 and 3.8 times that for a palmar angle of 0°. Stored elastic energy at the distal laminar junction border was small in all cases. For the breakover case, stored elastic energy at the proximal border also increased with increasing palmar angle. Conclusions and potential relevance: The models in this study predict that raising the palmar angle increases the load on the dorsal laminar junction. Therefore, hoof care interventions that raise the palmar angle in order to reduce the dorsal lamellae load may not achieve this outcome. See also correspondence by Redden See also correspondence by Curtis     

Congenital hypomyelinating polyneuropathy in two golden retriever littermates

Serial peripheral nerve biopsies from two golden retriever littermates with chronic neurologic disease were taken for morphologic and morphometric evaluation. Teased nerve preparations were difficult to interpret due to the lightness of myelin staining. Light and electron microscopic findings were characterized by the following: reduced number of myelinated axons, presence of myelinated sheaths inappropriately thin for the caliber of the fiber, poor myelin compaction, increased numbers of Schwann cell nuclei, increased concentration of neurofilaments in myelinated axons, many Schwann cells with voluminous cytoplasm, and increased perineurial collagen. Onion bulb formation was not seen. In contrast to control data, a poor correlation was seen between numbers of myelin lamellae (ML) and axonal circumference (AC). The frequency distribution of ML ranged from 5 to 55 lamellae in affected animals (mean, 28 lamellae) compared to 20 to 140 lamellae in controls (mean, 66 lamellae). The ML/AC ratio was significantly reduced (P less than 0.001) in nerves of affected dogs. Morphometric results indicated that fibers of all calibers were hypomyelinated.     

On the development of the papillary body in the feline claw

The pre- and post-natal development of the feline claw was studied in 22 feline fetuses with a crown-rump length (CRL) ranging from 40 to 160 mm, in six kittens up to an age of 22 days, and in four adult cats. In fetuses up to a CRL of 75 mm, the characteristic shape of the feline claw was developed. Segment-specific dermal modifications in the various segments, especially the dorsal ridge, started to develop in fetuses with a CRL between 75 and 105 mm. Modifications of the papillary body in the different claw segments took place in the last third of prenatal development and were continued postnatally. At first, the basal lamina became wavy, followed by the formation of small dermal microridges, which would be enlarged to dermal ridges and lamellae. In the claw of adult cats, the papillary body was very small. The dermal tissue of the proximal part of the coronet formed low microridges with short papillae originating on and between these low ridges. In the wall segment, dermal microridges were formed which were arranged in a parallel fashion, and in the distal part of the wall, short dermal micropapillae arose on the crest of each microridge. In the sole segment, thin dermal lamellae were developed. The sequence of papillary body development and the varying conformations of the papillary body in the different segments of the feline claw are compared to those in the nail, the canine claw and hooves.     

Scanning-Electron Microscopy of the Development of the Head of Rainbow-trout (Oncorhynchus mykiss) Embryos

The development of the cephalic region of rainbow trout in the 24th, 30th and 36th stages, corresponding to the table of development by Vernier (1969), was studied using light microscopy and SEM. The 24th stage shows a voluminous cephalic region as a principal feature. At this time, most components of this region have started their differentiation. The 30th stage can be considered as an intermediary phase in the embryonic formation, and it is characterized by the high degree of differentiation between the nervous system and the sense organs. In the 36th stage, the basic final shape of the embryo has been reached. The results of this study show that the differentiation of gill lamellae and the opening of the mouth takes place later than that moment described by other authors. This suggests that development between 30th and 36th stages is a critical survival period for rainbow trout, in which respiratory and digestive functions start.     

PEG胁迫对经60Co-γ辐射无芒雀麦种子的幼苗叶片超微结构的影响

为明确⁶⁰Co-γ射线对干旱条件下无芒雀麦（Bromus inermis）叶片的影响,从而为无芒雀麦利用辐射进行抗旱育种提供理论依据,本试验应用透射电镜观察聚乙二醇（Polyethylene glycol,PEG）模拟干旱胁迫下的⁶⁰Co-γ射线辐射无芒雀麦的叶片细胞,比较辐射对无芒雀麦叶片细胞、叶绿体、线粒体的形态和超微结构的影响。试验结果表明：未受辐射和PEG胁迫的无芒雀麦叶片细胞形态规则,叶绿体基粒片层和基质片层多且有序,线粒体膜完整,内嵴清晰,基质浓厚。10% PEG胁迫50 Gy辐射处理组叶片细胞扭曲最严重,形态不规则,细胞壁厚度不均,质膜内陷,有轻微质壁分离。10%和20% PEG胁迫下辐射剂量为50 Gy时,叶绿体和线粒体形态和超微结构受影响程度较轻,随着干旱胁迫强度和辐照剂量的增加,叶绿体和线粒体超微结构受损伤程度增强;20% PEG胁迫下辐射剂量为200 Gy时,叶绿体膜有不同程度破损,基粒片层排列的紊乱性增加,部分基粒片层不清晰,基质类囊体片层结构清晰,可见少量淀粉粒;线粒体膜破损比较严重,内嵴减少,排列不规则,基质较浅。可见,不同⁶⁰Co-γ射线辐射剂量可对无芒雀麦叶片叶绿体和线粒体超微结构产生不同程度的影响,在利用⁶⁰Co-γ射线辐射进行无芒雀麦耐旱性育种时,可选择50 Gy低辐射剂量结合20% PEG模拟干旱胁迫进行抗旱性突变体的筛选。     

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.     

氮酮促进洛美沙星仔猪皮肤吸收机理的研究

选用10～12日龄同品种仔猪,随机分组,于仔猪腹部皮肤涂搽含氮酮的洛美沙星透皮吸收剂, 分别于涂药前、涂药后2、4、6、12、24、48和72 h 在体取皮,应用透射电镜观察皮肤表皮结构变化,探讨氮酮促进洛美沙星透皮吸收的作用机理.结果表明,氮酮通过以下途径促进药物透皮吸收:(1)作用于表皮角质层细胞间脂质,使角质层变得疏松,细胞间距增大,外角质层细胞易于脱落,降低了皮肤对药物的屏障作用;(2)氮酮进入角质细胞内,与细胞内基质相作用,引起基底角质层肿胀,增加了角质细胞水化程度和药物存储空间.研究还发现,氮酮在体对仔猪皮肤的作用可维持72 h 以上;由氮酮引起皮肤结构的改变是一非损伤可复性过程.     

梅花鹿角的生药学研究

本文对药材梅花鹿角进行了生药学方面的研究。结果表明 ,梅花鹿角具有独特的性状特征 ,其公式为 A1P1,A2 P2 ,A3P3。梅花鹿角的显微结构由颓废层、骨密质和骨疏质组成。颓废层均有向内凹陷的窝和管。骨密质由哈佛氏系统组成 ,观察到了暗色带。骨疏质的网眼及骨小梁上首次发现棕色球形物 (直径 1 5～ 50 μm)。粉末中有菊花状等不同形状的骨板碎片、骨疏质碎片和颓废层碎片 ,及成群的棕色球形物