Alopecia areata in C3H/HeJ mice involves leukocyte-mediated root sheath disruption in advance of overt hair loss

Alopecia areata (AA) can be induced in C3H/HeJ mice by grafting full-thickness AA-affected skin. An 8- to 12-week delay between surgery and overt hair loss onset provides an opportunity to examine disease pathogenesis. Normal haired C3H/HeJ mice were sham-grafted or grafted with AA-affected skin. Mice were euthanatized 2, 4, 6, 8, 10, and 12 weeks after surgery along with chronic AA-affected mice as a positive control. Until 6 weeks after grafting, inflammation was only evident around anagen-stage hair follicles in host skin adjacent to but not distant from the AA-affected graft. From 8 weeks on, AA-grafted but not sham-grafted mice exhibited a diffuse dermal inflammation at distant sites that progressively focused on anagen-stage hair follicles at 10 and 12 weeks. Perifollicular inflammation was primarily composed of CD4+ and CD8+ cells associated with follicular epithelium intercellular adhesion molecule -1 expression. Only CD8+ cells penetrated intrafollicularly by 12 weeks after surgery, although both CD4+ and CD8+ intrafollicular cells were observed in chronic AA-affected mice. Under electron microscopy, intrafollicular lymphocyte and macrophage infiltration associated with hair follicle dystrophy was prominent 10 weeks after surgery, primarily within the differentiating outer and inner root sheaths. This study shows that focal follicular inflammation develops some time in advance of overt hair loss and focuses on the differentiating root sheaths in C3H/HeJ mice. The severity of inflammation and the degree of hair follicle dystrophy induced by the infiltrate appear to reach a threshold level before overt hair loss occurs.     

幼年牦牛不同部位皮肤毛囊的组织学结构及TGF-β2与HIF-1α差异性表达分析

旨在探究幼年牦牛皮肤毛囊的组织学结构和TGF-β2及HIF-1α对幼年牦牛皮肤毛囊生长发育的影响，选取10头健康幼年牦牛，采集其颈部、背部、胸部、腹部、小腿部、腋下及阴囊皮肤组织，制作石蜡切片后，采用HE和Sacpic染色，对各部位皮肤组织中毛囊进行观察和计数，并筛选出多毛及少毛部位。利用qRT-PCR、Western blot及免疫组织化学法对TGF-β2和HIF-1α在幼年牦牛多毛皮肤和少毛皮肤进行定位与定量的初步研究。结果表明，幼年牦牛皮肤的毛囊分布于真皮层，常与汗腺及皮脂腺伴行，毛髓质及内根鞘结构不完整。Sacpic染色可见毛囊内根鞘为红色，外根鞘为苍绿色，结缔组织鞘为蓝绿色。腹部皮肤的毛囊数量最多[（2 085±15）个·cm^-2]，阴囊数量最少[（158±15）个·cm^-2]。免疫组织化学结果显示，TGF-β2及HIF-1α主要表达在表皮层、毛囊外根鞘、皮脂腺及汗腺。TGF-β2在阴囊和腋下的mRNA转录水平和蛋白质表达水平均显著高于腹部和背部。HIF-1α在腹部的mRNA转录水平和蛋白质表达水平均显著高于其他三个部位。幼年牦牛的毛囊处于退行期，在腹部数量最多，背部次之，阴囊最少；TGF-β2和HIF-1α在不同部位皮肤中的表达水平存在显著差异，为进一步研究TGF-β2和HIF-1α对牦牛皮肤毛囊生长发育的影响提供理论依据。     

Effects of Photoperiod on Stem Cells Activation in Cashmere Goat Hair Follicles

In order to extend the anagen of cashmere goat hair follicles and increase the production of cashmere,this study was performed with artificially shorten the daylight time among Arbas White cashmere goats. Skin tissue sections from cashmere goats were collected to compare the morphologic changes between artificial daylight and natural daylight,and immunohistochemical method was used to study the hair follicle cell proliferation and important protein expression in related signaling pathways. The results showed that strong cell proliferation occurred in cashmere goat hair follicle cells during artificial daylight,plenty of cytokeratin 15 (K15) positive signals were distributed in the outer root sheath,β-catenin protein was actively expressed in hair matrix and root sheath, indicating that the hair follicles were in the anagen growth phase;Meanwhile,cashmere goat hair follicles under natural daylight were in telogen with weak signals. Above all prove that short photoperiod played an important role in promoting hair follicle growth,the artificial short photoperiod could change hair follicle growth cycle and make hair follicles earlier enter to the anagen growth phase,causing a variety of typical gene expressions during hair follicle growth.     

光周期对于绒山羊毛囊干细胞激活的影响

为了延长绒山羊毛囊兴盛期,提高羊绒产量,本试验通过人工缩短内蒙古阿尔巴斯型绒山羊的日照时间,利用组织切片技术对比人工短光照和自然光照周期下绒山羊皮肤组织形态变化,利用免疫组织化学方法研究毛囊细胞增殖及相关信号通路重要蛋白的表达。结果显示,人工短光照周期下绒山羊毛囊细胞增殖强烈,干细胞标记角蛋白15（cytokeratin 15,K15）阳性信号大量分布于外根鞘,β-catenin信号活跃表达于毛母质及根鞘,毛囊提前进入兴盛期;而自然光照周期下绒山羊毛囊仍停留在休止期,相应蛋白信号表达较弱。综上表明,短日照对毛囊生长具有明显的促进作用,人工短光照周期可以提前激活毛囊干细胞,使其提前进入兴盛期,引起多种毛囊生长相关蛋白的表达。     

水貂毛皮成熟期皮肤中EGF、IGF-Ⅰ及IGF-ⅠR基因的表达

为探讨水貂皮肤和毛囊发育机理,试验运用组织学和免疫组化方法对水貂毛皮成熟期皮肤及毛囊中表皮生长因子（epidermal growth factor, EGF）和胰岛素样生长因子-Ⅰ（insulin-like growth factor-Ⅰ,IGF-Ⅰ）及其受体（insulin-like growth factor-Ⅰreceptor,IGF-ⅠR）进行了研究。结果表明,水貂皮肤表皮细胞和毛囊外根鞘细胞中都有IGF-Ⅰ、IGF-ⅠR和EGF基因的强阳性表达,3种基因主要分布于细胞质中,细胞核呈空泡状未见到阳性染色,呈苏木精复染的蓝色,皱褶区域和表皮下胶原结缔组织出现了阳性染色为非特异性阳性结果。IGF-Ⅰ、IGF-ⅠR和EGF基因在水貂皮肤表皮和毛囊外根鞘细胞中广泛表达,直接参与调控皮肤和毛囊的发育。表明EGF、IGF-Ⅰ和IGF-ⅠR基因在水貂皮肤及其衍生结构的发育中发挥着重要作用。     

不同日龄滩羊皮肤组织化学特征比较

为比较2、35日龄滩羊皮肤毛囊的发育特点与血管内皮生长因子（vascular endothelial growth factor，VEGF）和血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）的分布特征，探究出生后滩羊被毛生长发育的变化特点，试验应用常规HE染色及改良Masson胶原纤维染色、Gomori银氨法染色、磷钨酸染色等特殊染色观察2与35日龄滩羊皮肤组织结构特点；应用免疫组织化学法结合免疫荧光染色法观察VEGF及VEGFR2在2与35日龄滩羊皮肤组织中的分布定位，并用IPP图像分析软件进行定量分析。结果显示：与2日龄滩羊皮肤组织比较，35日龄滩羊表皮与真皮间界限更加清晰，毛囊结构发育完整；毛囊密度显著降低（P<0.05）；胶原纤维与弹性纤维含量增加，形成网格状分布。免疫组化及免疫荧光结果显示，VEGF及VEGFR2在滩羊皮肤表皮及毛囊外根鞘、皮脂腺上均有表达。统计表明，VEGF及VEGFR2在2日龄滩羊皮肤组织中的表达量均显著高于35日龄（P<0.05）。综合上述结果，滩羊毛囊发育过程中，胶原纤维和弹性纤维增加明显；VEGF与VEGFR2通路在毛囊角质形成中起直接调节作用。     

Histology of the hair cycle in male beagle dogs

A detailed histologic study of the hair follicles of male Beagle dogs was made to determine changes in microscopic structure associated with the three stages in the hair cycle. Observed changes are that the bulbs of hair follicles in anagen are more closely associated with adipose tissue than they are in telogen. This is due to the deeper penetration of the follicles into the subcutaneous tissue during anagen. The hair germ cells of telogen were presumed to arise from the stratum basale of the matrix cells rather than from the outer root sheath cells. During telogen, the dermal papilla is separated from the club hair, but remains in close proximity to it. There is no connecting stalk as is reported for other animals.     

Notoedric mange in free-ranging masked palm civets (Paguma larvata) in Japan

Fifty-one masked palm civets (Paguma larvata) were trapped as part of a nuisance wildlife control programme between July 2001 and August 2002 in Fujisawa City, Kanagawa Prefecture, Japan. Eleven civets had characteristic mange lesions with marked alopecia and crusts, caused by the burrowing epidermal mite Notoedres cati. The diagnosis was confirmed by histology and examination of mites obtained from skin scrapings of affected civets. Histopathology of lesions demonstrated moderate hyperplastic epidermis, parakeratosis and acanthosis. Occasionally microvascular angiogenesis was observed in the epidermis. Tunnels were excavated in the hair follicles, reaching the hair roots. The tunnels were located between the hair shaft and the inner root sheath. Female mites, eggs and nymphs were demonstrated in their tunnels using scanning electron microscopy.     

内蒙古绒山羊Homeoboxc9基因在皮肤毛囊中的表达

试验通过原位杂交检测Homeoboxc9（Hoxc9）基因在皮肤毛囊中的表达情况,结果显示Hoxc9在胚胎期75 d的上皮细胞中表达,伴随着毛囊的生长发育,Hoxc9基因分别在初级毛囊的毛母质、毛乳头、内根鞘、外根鞘和毛干几个部位表达;在次级毛囊的内根鞘和绒干表达。在这个过程中毛囊角质化细胞大量增殖,表明在毛囊发育中Hoxc9基因对于角质化细胞的增殖可能起一定作用。     

Distribution of acid and alkaline phosphatases in canine skin.

Azo dye methods were used to determine the distribution of acid phosphatase (ACP) and alkaline phosphatase (ALP) in the skin of 25 Beagle dogs. ALP activity was found in dermal papillae of hair follicles regardless of their state of activity, in Huxley's layer of the inner root sheath of anagen stages, in myoepithelial cells of apocrine sweat glands, in germinative cells of sebaceous glands, in vascular endothelium, and in mast cells. The ACP activity was found in the epidermis, outer and inner root sheaths, keratogenous zone, hair cuticle and medulla, duct of sebaceous gland, and sebum. The results indicate that ACP and ALP are distinctive enzymes serving different biologic functions. The principal role of ALP in the skin appears to be dephosphorylation for adsorption and transport of chemical substances necessary for growth and maintainence of the pilary system and glandular adnexa. The ACP appears to be primarily involved in the breakdown of phospholipids and in necrobiosis of keratinocytes.     

催乳素对内蒙古绒山羊毛囊体外培养影响的研究

【目的】 研究催乳素（PRL）对内蒙古绒山羊初级毛囊和次级毛囊体外生长及形态变化的影响。【方法】 机械法结合切割法分离内蒙古绒山羊的初级毛囊和次级毛囊,在初级毛囊培养液中分别添加0、5、10、50、100 ng/mL催乳素进行体外培养,每组24根,共培养5 d,每天在显微镜下观察其形态并拍照,统计其生长长度、生长速度和存活率,筛选出最适催乳素处理浓度。然后将初级毛囊与次级毛囊分别分为初级毛囊对照组（PF-K）、初级毛囊试验组（PF-PRL）、次级毛囊对照组（SF-K）、次级毛囊试验组（SF-PRL）,每组24根,对照组用基础培养液培养,试验组在基础培养液中添加最适浓度的催乳素,培养5 d,每天观察毛囊的形态并拍照,同时测量各组毛囊的生长长度。【结果】 10 ng/mL催乳素组毛囊的平均日生长长度均极显著高于其他浓度组（P<0.01）,最终生长长度和存活率均最高,因此,后续试验选择10 ng/mL催乳素处理毛囊。试验组和对照组初/次级毛囊的毛干与根鞘部位同时伸长,随着培养时间的增加均出现不同程度的弯曲。PF-PRL、SF-PRL组毛囊在2~5 d的总长度分别极显著高于PF-K、SF-K组（P<0.01）。PF-K组除第1天与第0天差异不显著外,1~5 d毛囊的总长度依次显著增加（P<0.05）;PF-PRL组0~5 d毛囊的总长度依次显著增加（P<0.05）。SF-K组毛囊第5天的总长度显著高于0~4 d （P<0.05）;SF-PRL组第4、5天毛囊的总长度均显著高于0~3 d （P<0.05）,第3天毛囊的总长度显著高于0~2 d （P<0.05）。PF-PRL、SF-PRL组毛囊在2~5 d的平均日生长长度分别极显著高于PF-K、SF-K组（P<0.01）。【结论】 10 ng/mL催乳素是体外促进毛囊生长的最适浓度,10 ng/mL催乳素对体外培养的内蒙古绒山羊的初级毛囊和次级毛囊均有极显著的促生长作用。     

CD34 glycoprotein identifies putative stem cells located in the isthmic region of canine hair follicles

It is widely documented that a pool of multipotent stem cells located in humans and mice hair follicle outer root sheath (bulge region) is involved in the restoration of the whole follicular unit during each anagen phase. To the authors' knowledge, data regarding the location and characterization of hair follicle stem compartment in dogs have not been reported in the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor cell antigen CD34 as a marker of putative stem cells located in a bulge-like region of canine hair follicles. The presence of CD34 mRNA and glycoprotein was assessed on formalin-fixed, paraffin-embedded canine skin samples by in situ hybridization technique and by standard immunohistochemistry, respectively. A strong expression of CD34 mRNA and glycoprotein was observed in a well-defined area of the hair follicle isthmic region and appeared uniformly concentrated at the level of the basal layer of the outer root sheath. These findings provide compelling support to the hypothesis that in dogs, a subpopulation of basal keratinocytes located in the hair follicle isthmic region and characterized by the selective expression of CD34 is potentially associated with the stem cell compartment of this skin appendage.     

Cell proliferation kinetics in the hair root matrix of dogs with healthy skin and dogs with idiopathic seborrhea

Cell proliferation kinetic values were established for the hair root matrix of primary anagen follicles of 14 Beagles and 4 Cocker Spaniels with healthy skin and 9 Cocker Spaniels with primary idiopathic seborrhea. Indices were established by intradermal pulse labeling with tritiated thymidine, followed by cutaneous biopsy and autoradiography. The hair root matrix cell labeling index was 23.4 +/- 3.5% for Beagles, 24.4 +/- 4.0% for healthy Cocker Spaniels, and 24.9 +/- 4.3% for seborrheic Cocker Spaniels. These values indicate a rapidly proliferating cell population. Differences among these cell kinetic data for the 3 groups of dogs were not statistically significant. Although significant cell kinetic differences have been reported for other epidermal structures (interfollicular epithelium, upper hair follicle external root sheath, sebaceous glands) in seborrheic Cocker Spaniels, proliferation of hair root matrix cells apparently remains unaffected.     

MC1R蛋白在不同毛色太行山羊皮肤组织中的表达定位

MC1R基因是调节哺乳动物毛色形成的重要候选基因之一,编码黑色素皮质素受体1（MC1R）,在哺乳动物黑色素的代谢中发挥重要作用。分别选取纯黑色、纯白色、黄褐色和黑斑白色的太行山羊皮肤组织,通过HE染色技术,观察太行山羊的皮肤组织结构;利用免疫组化技术,对MC1R蛋白在4种毛色太行山羊皮肤组织中的表达情况进行研究。结果表明,太行山羊皮肤由表皮、真皮、皮下结缔组织3部分组成;MC1R蛋白在4种毛色太行山羊的表皮、真皮中均没有表达,但在纯黑、黄褐和黑斑白色太行山羊毛囊中有表达,且主要集中在毛囊外根鞘处。研究结果为太行山羊进一步的提纯选育工作提供了一定的依据。     

Microscopic anatomy of the skin of the woodchuck (Marmota monax): comparison of woodchuck hepatitis virus-infected and non-infected animals.

Thirty-three woodchucks were used in this study. Seventeen animals were healthy adults, not infected with woodchuck hepatitis virus (WHV); 10 were healthy adults infected with WHV; 4 were noninfected neonates; 2 were infected neonates. Within the 4 groups of woodchucks, no histologic differences were detected on the basis of sex or age. Neither were histologic findings different between infected and noninfected woodchucks of similar ages. The average thickness of skin (as measured from the skin surface to the inner limit of the dermis) from the general haired body area was 2394 microns. The skin was thickest on dorsal body areas, and gradually became thinner on ventral body and medial limb areas. The epidermis consisted of 4 layers: stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. A stratum lucidum was present only in the epidermis of the footpads. There was no clear distinction between the superficial dermis and the deep dermis, except for the subtle differences in arrangement and size of collagen fibers. Elastic fibers were seen throughout the dermis, being more prominent in the superficial portion. Both compound and simple hair follicle arrangements were seen, with compound being more common. The arrectores pilorum muscles were largest in the skin over the dorsal body areas. Sebaceous glands were present either within the outer root sheath of hair follicles or in the dense connective tissue surrounding hair follicles. No apocrine sweat glands were found. However, there were abundant eccrine sweat glands in the subcutaneous fat of the footpads.     

Proliferative and necrotizing otitis externa in four cats

Proliferative and necrotizing feline otitis externa is a rare disorder of unknown aetiology. This condition was diagnosed by skin biopsy in three adult domestic shorthair cats (3-5 years old) and one kitten (6 months old). The affected cats had large tan to dark brown-black coalescing plaques covering the concave surface of the pinnae and external ear canals. Friable material from the plaques and a thick exudate occluded the ear canals. The cats had a secondary bacterial and/or yeast otitis. Prior to the histopathological diagnosis, all cats received numerous otic preparations as well as oral antibiotics and corticosteroids without resolution. Histologically, all cases had strikingly similar changes; acanthosis with pronounced hair follicle outer root sheath hyperplasia and neutrophilic luminal folliculitis, follicular keratosis and individually necrotic keratinocytes in the outer root sheath of hair follicles. One case was documented via skin biopsy to have persisted for 4 years. The adult cats were treated with topical 0.1% tacrolimus and all showed marked improvement although one cat was lost to follow up. The lesions completely resolved with topical tacrolimus alone in one cat and topical tacrolimus in addition to oral prednisolone in another cat.     

Immunohistochemical localization of fibroblast growth factor 18 in hair follicles of healthy beagle dogs

Increasing emphasis is being placed on the role of fibroblast growth factors (FGFs) in hair follicle cycling. In mice, expression of FGF18 mRNA peaks during the late telogen phase, leading to the hypothesis that FGF plays a role in anagen induction. There are no data on the presence of FGF18 in dogs. The main objective of this study was to identify and locate FGF18 in the canine hair follicle. The second objective was to assess potential differences in FGF18 concentration between biopsies taken in winter and summer, shoulder and flank regions, and between different sexes. Skin tissue from 10 healthy beagle dogs (three intact females, three spayed females and four intact males) was collected from the shoulder and flank. The biopsies were collected in February and August on day 0, after which the dogs were clipped and biopsies collected again from the shoulder and flank on days 1, 3, 7 and 17. Paraffin sections (4 μm thick) of the biopsies were stained with an anti-FGF18 antibody. The FGF18-positive cells were counted in the hair follicle epithelium from seven follicular units of each biopsy. Fibroblast growth factor 18 was detected as granular cytoplasmatic staining in follicles at the level of the inner root sheath, and rarely in the outer root sheath and dermal papilla. It was also detected in the apocrine glands, in arrector pili muscles and in vascular endothelial cells. There was no statistical difference in the number of FGF18-positive cells or follicles between sexes, different anatomical locations, seasons or the consecutive days of sampling.     

Prenatal development of adenohypophyseal cell types, ovary, and uterus of dromedary camel

The somatotrophic and thyrotrophic cells were originally developed in the adenohypophysis from undifferentiated chromophobic cells at the foetal age of 4-8 weeks. The follicle-stimulating hormone cells appeared at a foetal age of 24-28 weeks. Prolactin and luteinizing hormone cells were first seen at of foetal age levels of 32-36 and 40 weeks. The germinal epithelium of flattened and cuboidal cells in the ovary was changed to cubical and columnar types, 24-28 weeks of foetal age. The sex cords appeared at 8-12 weeks of foetal age. The primary and secondary follicles were observed at 8-12 and 20-24 weeks of foetal age, respectively. Follicular cavity was noticed at 32-36 weeks, while large follicles encapsulated by follicular sheath were seen in the 40th week of foetal age. Some ovarian follicles degenerated during or after their development. Surface epithelium in the uterus changed from cubical to columnar type at 8-12 weeks of foetal age. The endometrium was clearly distinguished from the myometrium at 16-20 weeks of foetal age. The uterine glands appeared at 16-20 weeks as short and straight tubular glands, and they began to branch in the 40th week of foetal age.     

The effects of thyroid hormones on the skin of beagle dogs

The effects of hypothyroidism on canine skin were determined by comparing morphologic, morphometric, and hair cycle differences in skin biopsy samples from 3 groups of age- and gender-matched Beagle dogs: (1) euthyroid dogs; (2) dogs made hypothyroid by administration of 131I; and (3) dogs made hypothyroid and maintained in a euthyroid state by treatment with synthetic thyroxine. After 10 months of observation, there was slower regrowth of hair 2 months after clipping in the untreated-hypothyroid dogs. Untreated-hypothyroid dogs had a greater number of follicles in telogen and fewer hair shafts (ie, a greater number of hairless telogen follicles) than did the control group. The control dogs had a greater number of telogen follicles but the same number of hair shafts as the treated-hypothyroid group. Treated-hypothyroid dogs had the greatest number of follicles in the growing stage of the hair cycle (anagen). This study suggests that, at least in Beagles, induced hypothyroidism does not affect the pelage as dramatically as has been described in naturally occurring disease. This is because normal Beagles retain hair shafts in follicles for long periods, and the alopecia of hypothyroidism appears to evolve slowly because of the prolongation of this haired telogen stage. The evaluation of thyroxine-treated hypothyroid dogs demonstrates that thyroid hormone supplementation of Beagle dogs with induced hypothyroidism stimulates hair growth.     

Decrease of nuclear reactivity to growth-regulatory galectin-1 in senescent human keratinocytes and detection of non-uniform staining profile alterations upon prolonged culture for galectin-1 and -3

Summary Multipotent stem cells (source for interfollicular epidermis, hairs and sebaceous glands) are localized in the bulge region of the outer root sheath of hair follicles, while stem cells giving rise to interfollicular epidermis reside in its basal. Using the multifunctional lectin galectin-1 as a marker to localize accessible binding sites in situ as a step to figure out galectin functionality in stem cells, we studied hair follicle-derived keratinocytes. Specific nuclear binding of galectin-1 associated with expression of DeltaNp63alpha, a potential marker of epidermal stem cells, was detected. Binding of chimera-type galectin-3 to a nuclear site was not found in parallel assays. During the process of ageing in culture when cells acquire properties of senescence, disappearance of the nuclear signal for galectin-1 binding was accompanied by a similar decrease of nuclear DeltaNp63alpha expression and increased binding of galectin-3 to the cell membrane, namely in regions of intercellular contacts. Expression of cytokeratin 10, a marker of the terminal differentiation was seen only in a small fraction of the cell population. These data extend the evidence for nuclear sites with galectin-1 reactivity in squamous epithelial cells, the expression of which is modulated upon senescence. Moreover, the results document the divergence of galectin-1 and -3 on the level of ligand selection in this cell type, underscoring the importance of the technical aspect to employ tissue lectins as probe and to perform a fingerprinting with several markers of the galectin family in parallel.