特应性皮炎患犬的上表皮层在功能和结构上的变化

王帆

The upper epidermis of atopic dogs is altered at the functional and structural levels

特应性皮炎患犬的上表皮层在功能和结构上的变化

作者：Daniel Combarros, Dominique Goudouneche, Marie-Christine Cadiergues, and Michel Simon

翻译：王帆

Background – The pathogenesis of human atopic dermatitis (AD) is complex. Like humans, dogs develop spontaneous AD so this species could be a useful model of study. However, AD has been less characterised in dogs than in humans.

Objectives – To compare the epidermis of normal and spontaneously atopic dogs at the functional and structural levels.

Animals – Six healthy and five atopic laboratory Beagle dogs.

Methods and materials – Dogs were clinically characterised by general examination, Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04) evaluation and trans-epidermal water loss (TWEL) measurement. Skin biopsies were taken from healthy skin from normal dogs and on nonlesional and lesional skin from atopic dogs. Samples were analysed using transmission electron microscopy (TEM). Cornified envelopes were extracted and examined for their visual aspects (smooth versus ruffled).

Results – CADESI-04 and TWEL were significantly higher in atopic dogs. Healthy and nonlesional skin could be distinguished from lesional skin by histopathological evaluation. TEM examination revealed abnormal morphology of the stratum corneum (SC) in atopic skin. The SC compactum corneocyte layer was larger. Thicker and wrinkled corneocytes were more prominent (P = 0.005) in the lesional skin. Similar changes were observed in the nonlesional skin, but less pronounced. The proportion of immature ruffled envelopes was increased in atopic samples (P < 0.05), both from lesional and nonlesional areas.

Conclusions – The morphology of the SC was altered in the lesional and apparently nonlesional skin of spontaneously atopic dogs.

摘要

背景 – 人特应性皮炎(AD)的发病机制复杂。与人类一样, 犬也会出现自发性AD, 因此该物种可能是有用的研究模型。然而, 犬中的AD特征少于人。

目的 – 在功能和结构水平上, 对比正常犬和自发性特应性皮炎患犬的表皮层。

动物 – 6只健康犬和5只实验室特应性皮炎比格患犬。

方法和材料 – 通过全身体格检查、犬特应性皮炎严重程度指数、第4版(CADESI-04)评价表和经皮失水量(TWEL)对犬进行临床表现评估。对健康犬正常皮肤和特应性皮炎患犬的非病变皮肤和病变皮肤进行皮肤活检采样。 使用透射电子显微镜(TEM)分析样品。提取角化包膜并检查其外观(平滑与褶皱) 。

结果 – 在特应性皮炎患犬中CADESI-04和TWEL显著较高。通过组织病理学评估，健康和非病变皮肤与病变皮肤有所不同。TEM检查发现特应性皮炎的皮肤角质层(SC)形态异常。SC角化细胞层的压实程度较高。在病变皮肤的角化细胞明显更厚更褶皱(P = 0.005), 在非病变皮肤中也有此倾向，但不明显。在特应性皮炎病变皮肤和非病变皮肤样本中，未成熟皱褶角化包膜的比例都明显增加(P < 0.05)。

结论 – 在自发性特应性皮炎患犬的病变皮肤和看上去无病变的皮肤中，SC的形态都发生了改变。

Introduction

介绍

Atopic dermatitis (AD) is a common chronic relapsing skin disease in Western countries with a complex and not fully understood pathogenesis involving primary and secondary epidermal barrier defects together with an abnormal T-helper (Th)2 and Th22 immune response most commonly directed towards environmental allergens. A defective skin barrier is increasingly considered as the disease trigger leading to epidermal permeability and percutaneous allergic sensitisation.

特应性皮炎(AD)是西方国家一种常见的慢性复发性皮肤病，其发病机制复杂且尚未完全了解，包括原发性和继发性表皮屏障缺陷以及T辅助细胞(Th)2和Th22的异常免疫反应，这种反应通常是针对环境过敏原。皮肤屏障缺陷越来越被认为是疾病经皮渗透和经皮致敏的的诱因。

Dogs are a promising animal model, because they can develop a chronic allergic skin disease mirroring human AD. Shared characteristics include similar prevalence, similar lesional distribution and increased trans epidermal water loss (TEWL) suggesting epidermal barrier alterations.

犬适合做动物模型，因为它们出现的慢性过敏性皮肤病与人AD相似。共同特征包括相似的发病率、相似的病变分布和经皮失水量(TEWL)的增加，提示表皮屏障改变。

The purpose of this study was to compare functionally and structurally the epidermis of healthy and AD dogs.

本研究的目的是对比健康犬和AD犬表皮层的功能和结构。

Methods and materials

材料和方法

Ethics

道德准则

The study was approved by the Vivocore ethics committee (reference VRI185-18245-CO) and performed in accordance with the Animal for Research Act of Ontario and Canadian Council on Animal Care guidelines.

该研究由Vivocore伦理委员会批准(参考VRI185-18245-CO)，并按照安大略省和加拿大动物保护委员会的动物研究法案进行。

Animals

研究动物

Twelve beagle dogs owned by CanCog Technologies(Toronto, Ontario, Canada) and housed at Vivocore facility (Fergus, Ontario) were included in the study. Six dogs had been diagnosed with spontaneous AD, based on pre-defined diagnostic criteria and exclusion of other causes of pruritus. Six control dogs had no clinical history of AD, no other skin diseases, and no immunological impairment. All topical and systemic treatments in both groups were stopped at least one month before biopsies. All dogs were housed under the same conditions.

12只来自CanCog 科技的比格犬，在Vivocore实验室进行研究。根据预先诊断标准和排除其他瘙痒原因，6只犬被诊断为自发性AD。6只对照犬无AD临床病史，无其他皮肤病，无免疫缺陷。两组的所有外部和全身治疗至少在活检前一个月停止。所有的犬居住环境一致。

Lesion scoring and TEWL measurement

病变评分和TEWL测量

Clinical signs were scored using the Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04), and TEWL was measured using a closed chamber device (see Supporting information Appendix S1).

使用第4版犬特应性皮炎严重程度指数(CADESI-04)，使用闭式仪器测量TEWL(详见补充信息附录S1)。

Sample collection processing

采样过程

One 6 mm punch biopsy from normal abdominal skin was obtained from all dogs under general anesthesia with appropriate analgesia (see Supporting information Appendix S1). Additionally, one biopsy from lesional abdominal skin was sampled on four atopic dogs. Biopsies were halved with one half preserved in 10% formalin, and the other in 2% glutaraldehyde/Sorensen buffer.

在全身麻醉和适当的镇痛下，从所有犬的腹部正常皮肤上获得一个6mm打孔活检(见补充信息附录S1)。此外，对4只特应性皮炎患犬进行了腹部病变皮肤活检。活检样本一半保存在10%福尔马林，另一半保存在2%戊二醛/索伦森缓冲液中。

Histopathological evaluation included pattern analysis, and evaluation of inflammatory infiltrate and morphological changes associated with AD (Table S1).

组织病理学评估包括模式分析、炎症浸润评估和AD相关的形态学改变(表S1)。

Samples were reviewed blind with an electron microscope (HT7700, Hitachi; Tokyo Japan). Five stratum corneum (SC) microscopy fields were chosen at 91,000, 92,500, 95,000 and 915,000. The first and the second layers of granular cells were quantitatively analysed for: number of corneocyte layers in the SC compactum (SCC), number of granular keratinocyte layers, and number and size of keratohyalin granules (KHG). Corneocyte width at the level of the lower corneocyte layer was measured using IMAGEJ at five different areas on five images per sample. Average values were used for statistical analysis. KHG were delimited using IMAGEJ. The surface percentage corresponding to KHG was calculated by dividing the delimited area surface by the total surface of the first granular cell layer. Surface percentage was calculated in five fields from each sample and the average of the five measures was used for statistical analysis.

样本用电子显微镜。五张角质层(SC)显微镜下选择91,000,92,500,95,000和915,000。定量分析第1层和第2层颗粒细胞:致密SC (SCC)中的角化细胞层数、颗粒角质形成细胞层数、透明角质颗粒(KHG)的数量和大小。使用IMAGEJ在每个样本的5张图像上的5个不同区域测量下角细胞层水平的角细胞宽度。采用平均值进行统计分析。使用IMAGEJ分隔KHG。KHG对应的表面百分比是通过将划定的面积表面除以第一颗粒细胞层的总表面来计算的。从每个样品中计算五个区域的地表百分比，并使用五个测量值的平均值进行统计分析。

Cornified envelope examination

角化包膜检查

The method for examining cornified envelopes (CEs) is detailed in Appendix S1. Briefly, 10 μL of each sample were put on a slide, airdried and observed using phase-contrast microscopy to distinguish smooth (mature/rigid) from ruffled (immature/fragile) envelopes. In each case, 100 CEs were observed, and the ratio of smooth:ruffled structures was used for statistical analysis.

检验角化信封(CEs)的方法在附录S1中有详细说明。简单地说，每个样品取10 μL放在载玻片上，风干后用相差显微镜观察以区分光滑的(成熟的/坚硬的)和皱褶的(不成熟的/脆弱的)包膜。在每个病例中，观察到100个CE，并使用平滑:褶皱结构比进行统计分析。

Statistical methods

分析方法

Student’s t-test, ANOVA1, Wilcoxon–Mann–Whitney U-test and Kruskal–Wallis test were used, depending on the normality of data distribution, as detailed in Appendix S1. Statistical significance was set at P < 0.05.

根据数据分布的正态性，采用Student’s t检验、ANOVA1检验、Wilcoxon-Mann-Whitney u检验和Kruskal-Wallis检验，详见附录S1。P < 0.05有统计学意义。

Results

结果

One atopic dog (Case 10) displayed a CADESI-04 score of 9 on the day of sampling and was removed from the study.

1只特应性皮炎患犬(病例10)在取样当天CADESI-04评分为9分，被从研究中移除。

The control group (cases 1–6) comprised five females and one male, while the atopic group (cases 7, 8, 9, 11 and 12) comprised four females and one male. Mean age was 10.4 years (range 4–13 years) in the atopic group and 6.2 (range 4–10 years) in the control group (Figure 1a). The difference in age between groups was not statistically significant (P = 0.08, Wilcoxon–Mann–Whitney U-test). The CADESI-04 score was 5, on average, in the control group (range 2–10) and 21 (range 12–32) in the atopic group (P = 0.014) (Figure 1b).

对照组(病例1-6)雌性5例，雄性1例;特应性皮炎组(病例7、8、9、11、12)雌性4例，雄性1例。特应性皮炎组的平均年龄为10.4岁(4-13岁)，对照组的平均年龄为6.2岁(4-10岁)(图1a)。组间年龄差异无统计学意义(P = 0.08, Wilcoxon-Mann-Whitney u检验)。对照组CADESI-04评分平均为5分(范围2-10)，特应性皮炎组评分平均为21分(范围12-32)(P = 0.014)(图1b)。

The average TEWL value increased from 5.66 g/m2 /h in the control group, to 8.86 g/m2 /h in nonlesional atopic skin and 10.93 g/m2 /h in lesional skin (Figure 1c). The difference between control and lesional atopic skin was found to be statistically significant (P = 0.049, ANOVA1). Histopathological evaluation revealed no major differences between nonlesional atopic skin and healthy controls (Table S1 and Figure S1a–d). Lesional skin showed moderate to severe, focal to diffuse hyperkeratosis and acanthosis, perivascular to interstitial superficial mixed inflammatory infiltrates (lymphocytes, histiocytes, mast cells, plasma cells and fewer eosinophils), sebaceous gland hyperplasia and apocrine sweat gland dilation, with occasional hyperplastic epithelium (Table S1 and Figure S1e–h).

平均TEWL值从对照组的5.66 g/m2 /h增加到非病变性特应性皮炎皮肤的8.86 g/m2 /h和病变性皮肤的10.93 g/m2 /h(图1c)。对照组和病变性特应性皮炎皮肤之间的差异有统计学意义(P = 0.049, ANOVA1)。组织病理学评估显示，非病变性特应性皮炎皮肤与健康对照组之间无大差异(表S1和图S1a-d)。病变性皮肤显示中度至重度，局灶性至弥漫性角化过度和棘皮症，血管周至浅表间质混合性炎症浸润(淋巴细胞、组织细胞、肥大细胞、浆细胞和少量嗜酸性粒细胞），皮脂腺增生，顶浆汗腺扩张，偶见表皮增生(表S1和图S1e-h)。

Visualisation by TEM revealed alterations in the SC of atopic dogs (Figure 2). Corneocytes from healthy dogs (Figure 2a,b) showed a smooth and uniform surface with regular intercorneocyte spaces. The corneocyte thickness in the lowest layer was 0.34±0.05 μm (average±standard deviation). Corneocytes were tightly packed in the lower half of the horny layer forming the SCC, composed of 7±1.5 layers. In nonlesional atopic skin, the deepest corneocytes appeared irregularly shaped and wrinkled (Figures 2c-e). This finding was consistent and focally severe. Corneocytes tended to be thicker (0.58±0.18 μm, P = 0.06). The number of SCC layers was 8.3±4.1 and varied between different fields in the same skin. In some areas, the SCC was absent, and some wide intercellular spaces were observed in the deepest layers of the SC (Figure 2e); in others, it was thick with ≤16 layers of compacted corneocytes. In lesional skin the changes were more pronounced, with very thick corneocytes (0.78±0.2 μm; ANOVA1 P = 0.005) and a very thick SCC (9.27±0.47 layers) on average. The presence of marked intracorneocyte vesicles was observed (Figure 2f).

TEM显示特应性皮炎患犬SC的改变(图2)。来自健康犬的角化细胞(图2a,b)显示光滑均匀的表面，有规则的角化细胞间隙。最底层的角化细胞厚度为0.34±0.05 μm(平均标准差)。SCC角质层下半部紧密包裹角化细胞，形成7±1.5层。在非病变性特应性皮炎皮肤中，最深的角化细胞呈不规则形状和褶皱(图2c-e)。这一发现是一致且局部严重。角化细胞有增厚的趋势(0.58 ±0.18 μm, P = 0.06)。同一皮肤中SCC层数为8.3±4.1层，且相同皮肤不同视野间存在差异。在某些区域，SCC缺失，在SC的最深处可见一些宽的细胞间隙(图2e);在其他病例中致密角化细胞层数≤16层。病变皮肤的变化更为明显，有非常厚的角化细胞(0.78 ±0.2 μm;ANOVA1 P = 0.005)和平均非常厚的SCC(9.27 ±0.47层)。可见明显的角化细胞内囊泡(图2f)。

From healthy animal samples, smooth CEs (purified from skin biopsies) were predominant (average ratio smooth:ruffled CEs was 2.9), while from atopic samples, (both nonlesional and lesional areas) smooth and ruffled CEs were in more or less equal proportions (average ratios of 1.17 and 1.40, respectively; P = 0.03) (Figure 3).

在健康动物样本中，光滑CEs(从皮肤活检中提纯)占主导地位(光滑:皱褶CEs的平均比例为2.9)，而在特应性皮炎样本中(非病变和病变区域)，光滑和皱褶CE的比例基本相同(平均比例分别为1.17和1.40;P = 0.03)(图3)。

Focusing on the stratum granulosum (SG), the average number of granular cell layers was 1 in healthy skin, 1.2 in nonlesional atopic and 2.1 in lesional atopic areas (P = 0.02). The size, shape and numbers of KHG were highly heterogeneous between dogs in both groups (Figure 4), and no differences were observed when normal and atopic skin was compared. In particular, the proportions of the cytoplasmic surface corresponding to KHG in the most upper keratinocytes (SG1) were not statistically different: 6.6±6% in healthy skin, 5.91±2.9% in nonlesional skin and 8.58±2.6% in lesional skin. In the layer of keratinocytes just below (SG2), the KHG surface corresponded to a very low proportion of the cytoplasmic surface in normal and nonlesional skin (0.12% and 0.29%, respectively), and was increased in lesional skin (1.8%).

以颗粒层(SG)为例，健康皮肤颗粒细胞层平均为1层，特应性皮炎非病变皮肤颗粒细胞层数为1.2层，特应性皮炎病变皮肤颗粒细胞层数为2.1层(P = 0.02)。对比正常皮肤和特应性皮炎皮肤，两组犬的KHG的大小、形状和数量各种各样(图4)，对比后无差异。特别是最上层角质形成细胞上层(SG1)中对应KHG的细胞质表面比例无统计学差异:健康皮肤中为6.6%，非病变皮肤中为5.91±2.9%，病变皮肤中为8.58±2.6%。在角质形成细胞层(SG2)下方，在正常皮肤和非病变皮肤中，KHG表面对应的细胞质表面比例非常低(分别为0.12%和0.29%)，而在病变皮肤中则增加(1.8%)。

Discussion

讨论

In this study, TEM analysis revealed an abnormal appearance of the SC and corneocytes in both lesional and nonlesional skin of atopic dogs, as well as altered morphology of purified CEs. The SC presented irregular, wrinkled and thicker corneocytes, associated with a larger proportion of ruffled, immature CEs and increased TEWL, suggesting impaired cornification.

在本研究中，TEM分析显示特应性皮炎患犬的病变和非病变皮肤中的SC和角化细胞出现异常，纯化的CEs的形态也发生了改变。SC表现为不规则、皱褶和较厚的角化细胞，与较大比例的皱褶、不成熟的CE和TEWL增加有关，提示角化损伤。

At the ultrastructural level, the corneocytes of the SCC in nonlesional atopic skin appeared irregular, wrinkled and thicker, compared to the regular surface of control corneocytes. In parallel, an increased proportion of ruffled CEs was observed. This type of envelope is considered to be less mature and more fragile. To the best of the authors’ knowledge, these findings are novel. In two published studies, disappearance of the SCC was reported； this was not consistently observed in our study.

在超微结构水平上，非病变特应性皮炎皮肤中SCC的角化细胞与对照组正常的角化细胞相比呈现不规则、皱褶和更厚的表面。与此同时，观察到褶皱CE的比例增加。这种类型的包膜被认为是不成熟和更脆弱的。据作者所知，这些发现是新颖的。在两项已发表的研究中发现没有SCC，但在我们的研究中并没有一致发现。

Less abundant and abnormally organised intercorneocyte lipid lamellae have been described previously in dogs with AD. In the present study, the fixation method used allowed observation of corneocytes and not lipid lamellae. However, we observed vesicles in the matrix of corneocytes suggesting entombment of lamellar bodies, as already published in human and in an experimental model of canine AD, and abnormal lipid secretion.

以前曾报道过AD犬的角化细胞脂质层数量较少且组织异常。在本研究中，固定法能看到角化细胞，看不到脂质层。然而，我们观察到角化细胞基质中有囊泡，提示板层小体的埋没，正如在人类和犬AD实验模型中已经发表的那样，脂质分泌异常。

It could be hypothesised that the observed SC anomalies are primary defects because they were found in nonlesional skin without any apparent histological signs of inflammation. The abnormal quantity and distribution of intercorneocyte lipids in nonlesional skin, observed in other studies, also were considered primary events.

可以假设，观察到的SC异常是原发性缺陷，因为它们是在无病变皮肤中发现的，没有任何明显的组织学炎症表现。在其他研究中观察到的非病变皮肤中角化细胞间脂质数量和分布的异常也被认为是原发性问题。

Several study limitations may have limited data interpretation, including small number of samples (lesional skin), low CADESI-04 scores and the higher mean age of atopic dogs. However, as published previously, there was an inverse correlation between TEWL and the age of our normal dogs (R² = 0.448). Information is scarce on the influence of age on SC ultrastructural morphology. The SC structure appears conserved with age. In our samples, the abnormal structure of the SC was found in both young and old atopic beagles. Therefore, our findings were related to the atopic status and not age.

一些研究的局限性可能使数据解释受限，包括样本数量少(病变皮肤)、CADESI-04评分低以及特应性皮炎患犬的平均年龄较高。然而，如前所述，TEWL与我们正常犬的年龄呈负相关(R²= 0.448)。关于年龄对SC超微结构形态的影响的信息很少。SC结构随着年龄的增长而趋于保守。在我们的样本中，在年轻和年老的特应性皮炎比格犬中都发现了SC的异常结构。因此，我们的发现与特应性状态有关，而与年龄无关。

In conclusion, cornification is impaired in dogs with spontaneous AD. Lesional and nonlesional skin abnormalities in the SC, corneocytes and CE structure, and high permeability to water suggest involvement in pathogenesis. Further studies are needed to unravel the molecular background.

综上所述，自发性AD患犬的角化作用损伤。病变皮肤和非病变皮肤的SC、角化细胞和CE结构异常，以及高透水性提示参与了发病机制。需要进一步的研究来揭开分子背景。

haohao

秘鸢

南海

zhangenzyme

Vet武

青春年少