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Calcinosis cutis in dogs: histopathological and clinical analysis of 46 cases
46例犬皮肤钙质沉积症组织病理学及临床分析
翻译：王思权 校对：王帆
Background– Calcinosis cutis is well recognized in dogs with endogenous hyperglucocorticism and iatrogenic hyperglucocorticism, but the pathogenesis is still unclear.
Objectives– The objectives of the study were to identify possible correlations between histopathological patterns of dermal mineralization in skin biopsies and underlying causes for calcinosis cutis in dogs, as well as to determine breed predilection and age of onset for dogs within a hospital population. In addition, mineral analysis was performed on four biopsy samples.
Animals– Forty-six dogs with histopathologically confirmed calcinosis cutis were evaluated.
Methods- Medical records and histological sections of dogs with calcinosis cutis diagnosed by histopathology over a 21 year period were reviewed. Infrared spectrometry was used to identify the mineral in the paraffin blocks. Exact chi-squared test was used to identify breed predispositions, while a Mann–Whitney U-test was used to identify age correlations.
Results – Labrador retrievers, Rottweilers, boxers and Staffordshire terriers were the breeds most commonly affected in this study. Most dogs had either an exogenous or an endogenous source of corticosteroids, with the exception of five dogs with renal insufficiency. In the majority of cases, mineralization was found throughout the entire dermis. The average age of onset of calcinosis cutis for dogs with endogenous hyperglucocorticism was older than that of dogs with iatrogenic hyperglucocorticism. Using infrared spectrometry, apatite crystals were found to be the source of mineral.
Conclusions and clinical importance– There was no observable difference in the histopathological pattern of calcinosis cutis from dogs with endogenous hyperglucocorticism versus iatrogenic hyperglucocorticism. While glucocorticoid therapy appears to predispose dogs to developing calcinosis cutis, it remains unclear whether there is a specific dose or combination of factors that initiates the mineral deposition. Furthermore, the mineral deposition in dogs with calcinosis cutis was found to be apatite.
背景：在犬中，皮肤钙质沉积症被广泛认为是与内源性和医源性糖皮质激素增多症有关，但其发病机制尚不清楚。
目的：本研究的目的是确定皮肤活检中真皮矿化的组织病理学模式与犬皮肤钙质沉积症的潜在病因之间可能的相关性，并确定犬在医院病例种群中的品种倾向性和发病年龄。此外，对四个活检样本进行了矿物质分析。
动物：对46只经组织病理学证实为皮肤钙质沉积症的犬进行评估。
方法：本文回顾了21年来经组织病理学诊断为皮肤钙质沉积症的犬的病历和组织切片。用红外光谱分析鉴定了石蜡块中的矿物。精确卡方检验用于确定品种倾向，而Mann-Whitney U检验用于确定年龄相关性。
结果：本研究中最常见易患犬种是拉布拉多寻回犬、罗威纳犬、拳师犬和斯塔福梗。除了5只肾功能不全的犬外，大多数犬的皮质类固醇要么来自外源性，要么来自内源性。在大多数病例中，矿化涉及整个真皮层。内源性糖皮质功能增多症犬皮肤钙质沉积症的平均发病年龄大于医源性糖皮质激素增多症的犬。红外光谱分析表明，磷灰石晶体（apatite crystals）是皮肤矿化的来源。
结论和临床意义：内源性糖皮质功能增多症和医源性糖皮质激素增多症犬的皮肤钙质沉积症的组织病理学模式没有明显差异。虽然糖皮质激素治疗似乎使犬很容易患上皮肤钙质沉积症，但目前尚不清楚是特定剂量或是否有综合因素会引发矿物质在皮肤沉积。此外，在患有皮肤钙质沉积症的犬上发现的矿物沉积为磷灰石。
Introduction
概述
Calcinosis cutis (CC) is an uncommon occurrence, in which inorganic, insoluble mineral salts are deposited in the dermis, subcutis or, rarely, the epidermis. Most commonly, the mineral precipitation involves the collagen and elastin fibres in the dermis. This process can be seen secondary to, or in association with, a number of disorders. It is usually divided into the following four separate categories: metastatic, iatrogenic, dystrophic and idiopathic. Metastatic calcification describes calcium salts that precipitate within normal tissues as a consequence of underlying systemic calcium or phosphorous imbalance. This is most often seen in dogs in the context of impaired renal function, but has also been reported with systemic blastomycosis and paecilomycosis. Iatrogenic CC occurs secondary to percutaneous absorption or injection of calcium-containing products into the skin. Dystrophic calcification usually occurs as a result of local tissue injury and is seen most commonly in dogs with iatrogenic hyperglucocorticism or endogenous hyperadrenocorticism, but has also been reported in association with systemic disease (e.g. leptospirosis). Furthermore, conditions causing inflammation in the skin, such as follicular cysts, foreign body granulomas, interdigital pyoderma, demodicosis or pilomatrixomas, have also been reported to be associated with dystrophic calcification and ossification.
皮肤钙质沉积症(CC)是一种不常见的疾病，这是一种无机的、不溶性的矿物盐在真皮层、皮下组织或在表皮（罕见）沉积的疾病。矿物质沉积通常涉及真皮中的胶原和弹性蛋白纤维。这一过程可继发于其他疾病或与许多疾病相关。它通常分为以下四种不同的类型：转移性、医源性、营养不良性和特发性。转移性钙化描述了由于潜在的系统性钙磷失衡而在正常组织中产生钙盐沉积。这最常见于犬肾功能受损，但也有报道与系统性芽生菌病和拟青霉病有关。医源性CC继发于经皮吸收或向皮肤注射含钙产品。营养不良性钙化通常是局部组织病变的结果，最常见于患有医源性肾上腺皮质功能增多症或内源性肾上腺皮质功能增多症的犬，但也被报道与系统性疾病(如钩端螺旋体病)有关。据报道，引起皮肤炎症的疾病，如毛囊囊肿、异物肉芽肿、指间脓皮症、蠕形螨病或毛母质瘤，也与营养不良性钙化和骨化有关。
Dogs and chinchillas are the only known species of animals that develop CC in response to hyperadrenocorticism. Idiopathic CC has a histological appearance similar to glucocorticoid-induced CC. However, it occurs in dogs less than 1 year of age with no history of glucocorticoid excess and spontaneously resolves within a year. Idiopathic CC must be differentiated from calcinosis circumscripta, which also commonly occurs in young dogs but does not resolve spontaneously. Calcinosis circumscripta most commonly occurs on pressure points or areas of trauma, especially in young, growing dogs with enhanced calcium and phosphate metabolism. Histologically, calcinosis circumscripta is distinctive, with large, irregular foci of basophilic granular deposits that elicit a marked foreign body response and fibrosis.
犬和龙猫是唯一已知的因肾上腺皮质功能增多症而发展为CC的动物物种。特发性CC在组织学形态上与糖皮质激素诱导的CC相似。但是特发性CC发生于不到1岁、没有糖皮质激素过量使用史的犬中，并在一年内会自行消退。特发性CC必须与局限性钙质沉积症（calcinosis circumscripta）鉴别，局限性钙质沉积症也常发生于年轻犬中，但不会自发消退。局限性钙质沉积症最常发生在压力点或创伤部位，尤其是钙磷代谢旺盛的幼犬。局限性钙质沉积症的组织学是独特的，有大而不规则的嗜碱性颗粒状沉淀物，引起明显的排异反应和纤维化。
Metaplastic ossification in the skin, or osseous metaplasia, is characterized by deposition of calcium and phosphorous as hydroxyapatite crystals in a proteinaceous matrix, with formation of lamellar bone. While rare, it is typically seen in conjunction with multifocal dystrophic CC caused by iatrogenic hyperglucocorticism or endogenous hyperadrenocortism.
皮肤化生骨化或骨化生的特征是钙和磷在蛋白基质中以羟基磷灰石晶体的形式沉积，形成板层骨。虽然罕见，但它通常与医源性高糖皮质激素或内源性肾上腺皮质功能增多症引起的多灶性营养不良CC一起出现。
To the authors’ knowledge, no reports exist that review a large cohort of dogs with CC. As such, the aims of this study were to characterize patterns of mineralization and their association with other histological features and to identify possible correlations with underlying aetiologies. Potential breed, age and sex distributions were also investigated as possible predisposing factors of CC in dogs. In addition, analysis of the mineral composition of the calcified deposits was performed.
据作者所知，目前还没有大批量患有CC的犬的回顾性报道。因此，这项研究的目的是描述矿化的模式及与其他组织学特征的关联，并确定与潜在病因的可能的相关性。潜在的品种、年龄和性别分布也可能是犬CC的易感因素。此外，这篇文章对钙化沉积物的矿物成分进行了分析。
Materials and methods
材料和方法
Case selection
案例选择
The computer records of dogs seen at the Veterinary Medical Teaching Hospital, (VMTH) from 1 January 1989 to 1 August 2011 were searched using the keywords ‘calcinosis cutis’ and ‘canine’. Inclusion criteria were the diagnosis of CC via histopathology (diffuse mineral deposition in the dermal collagen and adnexal appendages).Dogs with calcinosis circumscripta, characterized by focal mineralization forming a mass, were excluded.
检索1989年1月1日至2011年8月1日在兽医教学医院(VMTH)所见的犬的计算机记录，关键词为“皮肤钙质沉积症”和“犬”。纳入标准是通过组织病理学(真皮胶原和其附属组织存在弥散性矿物质沉积)诊断CC。以局灶性矿化为特征的局限性钙质沉积症的犬被排除在外。
The signalment, body weight, age of onset of skin lesions, lesion distribution, serum calcium levels, concurrent disease and/or medical treatments and their duration were recorded from medical records.
从病历中记录其特征、体重、皮肤病变发病年龄、病变分布、血清钙水平、并发症和/或内科治疗及其持续时间。
Dogs were organized into the following four groups based upon the reported associated cause for CC: endogenous hyperadrenocorticism, suspect iatrogenic hyperglucocorticism, renal disease and unknown. For dogs with suspect iatrogenic hyperglucocorticism, the dose and length of the course of corticosteroids and/or administration of other immunosuppressive agents, as well as the disease for which corticosteroids had been prescribed, were recorded if this information was available.
根据已报道的CC相关病因，犬病例被分成以下四组：内源性肾上腺皮质功能增多症组、怀疑医源性糖皮质激素增多症组、肾脏疾病组和不明病因组。对于怀疑患有医源性高糖皮质激素的犬，如果有皮质类固醇和/或其他免疫抑制剂的治疗剂量和疗程长度、开具的类固醇药物的信息，也会被记录统计。
Histopathological review
组织病理学回顾
Haematoxylin and eosin stained sections of skin biopsies or postmortem skin samples from all dogs included in the study were reviewed. Two authors (V.K.A. and C.A.O.) were blinded as to the clinical history of the dog, while one author (K.A.D.) was not blinded. The following features were evaluated in each sample: (i) presence of epidermal atrophy; (ii) distribution of dermal mineralization, i.e. superficial (the dermo-epidermal junction to the sebaceous gland), mid-dermis (the sebaceous gland to the hair follicle bulb) or deep dermis (the hair follicle bulb to the subcutaneous tissue); (iii) pattern of mineralization, i.e.‘multinodular’ to describe multiple nodular mineral deposits, ‘multifocal’ for small, scattered mineral deposits and ‘diffuse’ for an even distribution of mineral throughout the dermis; and (iv) the presence and distribution of ossification.
对所有纳入研究的犬皮肤活检或死后皮肤样本的苏木素和伊红染色切片进行了回顾。其中作者V.K.A.和C.A.O.并不知晓研究中犬的临床病史，作者K.A.D.知晓。每个样本都进行了以下特征的评估：(i)是否存在表皮萎缩；(ii)真皮矿化的分布，即浅层(真皮-表皮与皮脂腺的交界处)、中层真皮(皮脂腺至毛囊球部)或真皮深层(毛囊球部至皮下组织)；(iii)矿化模式，即“多结节”指的是多个结节状沉积，“多灶性”指的是小而分散的沉积，“弥漫”指的是矿物质在真皮中均匀分布；(iv)骨化的存在和分布。
Additional histological findings recorded were as follows: (i) type of inflammatory cells; (ii) amount of fibrosis surrounding any mineral or bone, (iii) folliculitis and/or furunculosis; and (iv) transfollicular or transepidermal mineral elimination. The degree of inflammation, mineralization and fibrosis was assessed subjectively by three investigators as mild, moderate or severe and scored collectively.
记录的其他组织学发现如下：(i)炎症细胞的类型；(ii)任何矿物或骨周围的纤维化量；(iii)毛囊炎和/或疖病；以及(iv)经毛囊或经表皮的矿物消除率。炎症、矿化和纤维化的程度由三名研究人员主观评估为轻、中、重度，并进行集体评分。
Statistical analysis
统计分析
The breed distribution within the hospital population was evaluated using an exact chi-squared test, while the age distribution among iatrogenic hyperglucocorticism versus endogenous hyperadrenocorticism was evaluated using a Mann–Whitney U-test.
用精确卡方检验评估医院样本群的品种分布，用Mann-Whitney U检验评估医源性糖皮质激素增多症和内源性肾上腺皮质功能增多症患犬的年龄分布。
Mineral analysis
矿物分析
A total of four samples were examined for mineral extraction and evaluation, as follows: one from a dog with iatrogenic hyperglucocorticism, two from dogs with endogenous hyperadrenocorticism, and one from an unknown disease aetiology. Portions of the paraffin blocks of each case were melted, and the formalin-fixed tissue was collected in Eppendorff tubes.
总共有四个样本被用于矿物提取和评估，如下：一个来自医源性糖皮质激素增多症的犬，两个来自内源性肾上腺皮质功能增多症的犬，以及一个来自未知病因的犬。每个病例的石蜡包埋部分被熔化，福尔马林固定的组织被收集在Eppendorff试剂管中。
A razor blade was used to isolate tissue areas with large mineral deposits from the paraffin blocks. The processed samples were subsequently examined by the oil immersion method using a polarized-light microscope (Universal Polarizing Microscope, Carl Zeiss Inc., Thornwood, NY, USA) followed by infrared spectrometry to identify mineral components.
用剃刀从石蜡块中分离出有大量矿物质沉积的组织区域。随后使用偏光显微镜(Carl Zeiss Inc.，Thornwood，NY，USA)油浸法检查处理后的样品，然后用红外光谱鉴定矿物成分。
The potassium bromide disc method was adopted in infrared spectrometry. The processed sample and KBr powder were mixed together (approximately 1:150 w/w) and ground manually in an agate mortar with a pestle before the disc was pressed. The infrared spectra were collected using a Nicolet TMiS10 FT-IR spectrometer (Thermo Fisher Scientific Inc., Madison, WI, USA) at room temperature immediately after the preparation of the discs. Each spectrum was acquired by the accumulation of 36 scans at a resolution of 4 cm.
红外光谱分析采用溴化钾片法。将处理后的样品与溴化钾粉末混合(约1：150w/w)，在玛瑙砂浆中人工研磨，然后将圆片压制。用Nicolet TMiS10 FT-IR光谱仪(美国威斯康星州麦迪逊Thermo Fisher Science Inc.)在制备圆片后立即在室温下收集红外光谱。每种光谱通过以4厘米的分辨率累积36次扫描而获得。
Results
结果
The database search revealed 46 dogs that had been diagnosed with CC confirmed by histopathology with slides available for review.
数据库搜索显示， 46只犬经组织病理学证实被诊断为CC，并有可供回顾的组织切片。
Signalment
特征
Eight breeds of dogs represented over half of the cases in this study. The most common breeds were Labradors and Labrador mixed breeds (5 of 46; 10.8%), boxers and boxer mixed breeds (5 of 46; 10.8%) and Rottweilers (4 of 46; 8.6%). Akitas, Staffordshire terriers, golden retrievers, pit bull terriers, Pomeranians and German shepherd dogs each represented two of 46 or 4.3% of the cases in this study. Using an exact chi-squared test, there was a significant difference in the breed distribution of dogs affected with calcinosis cutis versus the rest of the VMTH population (P < 0.0001), with Rottweiler/Labrador retriever mixed breeds, Rottweilers, Staffordshire terriers, boxers, boxer mixed breeds, Akitas and Pomeranians over-represented. However, based on the small number within each breed, this finding should be regarded as tentative.
8个品种的犬代表了这项研究中一半以上的病例。最常见的品种是拉布拉多和拉布拉多混血品种(5/46，占10.8%)、拳师犬和拳师犬混血品种(5/46，占10.8%)和罗威纳犬(4/46，占8.6%)。秋田犬、斯塔福梗犬、金毛犬、比特斗牛犬、博美犬和德国牧羊犬每种占46例中的两例，占研究病例的4.3%。经卡方检验，患皮肤钙质沉积症犬的品种分布与其他犬种有显著差异(P<0.0001)，罗威纳/拉布拉多混血犬、罗威纳犬、斯塔福德郡犬、拳师犬、拳师混血犬、秋田犬和博美犬的比例过高。然而，基于每个品种的数量很少，这一发现应该被认为是不确定的。
Male dogs were the most common gender group represented (26 of 46), with castrated male dogs representing 36.9% (17 of 46) and intact male dogs 19.5% (9 of 46). Female spayed dogs represented 32.6% (15 of 46) and intact female dogs 10.8% (5 and 46). The median age at the time of diagnosis was 7.6 years (range 1–14 years).
性别倾向公犬最常见(26/46)，已去势公犬占36.9%(17/46)，未去势公犬占19.5%(9/46)。已绝育雌性犬占32.6%(15/46)，未绝育雌性犬占10.8%(5/46)。确诊时的中位年龄为7.6岁(1-14岁)。
The median age of the dogs with endogenous hyperadrenocorticism with CC was significantly higher (median 11 years; range 1–14 years) than the median age of dogs with iatrogenic hyperglucocorticism with CC (median 6 years; range1–12 years; P = 0.0005).
内源性肾上腺皮质功能增多症并发CC的中位年龄(中位数11岁；范围1~14岁)显著高于医源性高糖皮质激素并发CC的中位年龄(中位数6岁；范围1~12岁；P=0.0005)。
Physical examination
体格检查
The mean body weight of all dogs was 26.7 kg (range 2.4–59 kg). Dermatological findings varied from firm, palpable, erythematous papules to well-demarcated plaques. Most dogs had lesions in multiple regions of the body. The distribution of the lesions included the dorsum, flank, axilla, ventral thorax, extremities, inguinal region, head and perigenital region (see Table 1). The distribution of skin lesions had no apparent association with disease aetiology. The subjective nature of these observations in the medical records precluded the ability to perform statistical analysis.
全部犬体重范围是2.4~59 kg，平均26.7 kg。皮肤病的表现多样，从坚硬、可触摸的红斑丘疹到边界清楚的斑块。大多数犬在机体的多个区域都有病变。病变的分布范围包括背部、侧腹部、腋下、胸腔腹侧、指间、腹股沟、头部和生殖器周围区域(见表1)。皮肤病变分布与病原学无明显相关性。医疗记录中这些观察的主观性质不能够进行统计分析。
Medical history
病史
Upon review of medical records, potential underlying causes of the CC included iatrogenic hyperglucocorticism (25 of 46; 54.3%), endogenous hyperadrenocorticism (11 of 46; 23.9%), and renal insufficiency (6 of 46; 13.1%). Four dogs had unknown underlying disease. Hypercalcaemia was noted in three of 46 (6.5%) of the dogs for which biochemistry data were available for review. One hypercalcaemic dog was receiving calcitriol postparathyroidectomy and had concurrent chronic renal insufficiency [serum calcium 12.9 mg/dL (normal 9.6–11.2 mg/dL) and serum phosphorus 9.3 mg/dL (normal 2.6–5.2 mg/dL)], one hypercalcaemic dog had chronic renal insufficiency only (serum calcium11.8 mg/dL and serum phosphorus 13.5 mg/dL), and one hypercalcaemic dog was diagnosed with meningoencephalitis and was also treated with corticosteroids (serum calcium 13.5 mg/dL and serum phosphorus 13.1 mg/dL).
回顾病历，潜在CC的病因包括医源性糖皮质激素增多症(25/46，54.3%)、内源性肾上腺皮质功能增多症(11/46，23.9%)和肾功能不全(6/46，13.1%)。四只犬患有未知的潜在疾病。在有生化数据可供回顾的46只犬中，有3只(6.5%)发现有高钙血症。1只高血钙犬在甲状旁腺切除术后同时伴有慢性肾功能不全[血钙12.9 mg/dL(正常9.6~11.2 mg/dL)和血磷9.3 mg/dL(正常2.6~5.2 mg/dL)]，1只高钙犬仅有慢性肾功能不全(血钙11.8 mg/dL和血磷13.5 mg/dL)，1只高血钙犬确诊为脑膜脑炎，同时接受皮质类固醇治疗(血钙13.5 mg/dL和血磷13.1 mg/dL)。
 Known dosages of corticosteroids were recorded in 22 of 46 (47.8%) dogs, whereas in 12 of 46 (26.0%) the therapeutic history or dosages of corticosteroids were unknown; 12 of 46 (26.0%) dogs did not receive corticosteroids. Corticosteroids were the sole drug utilized in 11 of 46 (23.9%) dogs diagnosed with CC; azathioprine and corticosteroids were utilized in 10 of 46 (21.7%) dogs; ciclosporin and corticosteroids were used in five of 46 (10.8%) dogs; cyclophosphamide and corticosteroids were used in two of 46 (4.3%) dogs; and a combination of ciclosporin, azathioprine, corticosteroids and cyclophosphamide was utilized in one of 46 (2.1%) of dogs. Five dogs (10%) had other immunosuppressive/immunomodulatory medications along with oral corticosteroids, including vincristine, leflunomide, tetracycline/niacinamide, ophthalmic cyclosporine, and topical hydrocortisone. Diseases for which treatment with corticosteroids resulted in iatrogenic hyperglucocorticism were as follows: immune-mediated polyarthritis (4 of 22; 18.1%), immune-mediated haemolytic anaemia (3 of 22; 13.6%), myelopathy (3 of 22; 13.6%), meningoencephalitis (3 of 22; 13.6%), allergic dermatitis (2 of 22; 9.1%), pemphigus foliaceus (2 of 22; 9.1%), immune-mediated thrombocytopenia (2 of 22; 9.1%), aplastic anaemia (1 of 22; 4.5%), suspected cutaneous adverse drug eruption (1 of 22; 4.5%) and multifocal brain disease (1 of 22; 4.5%). Types of corticosteroids utilized were prednisone only (16 of 22; 72.7%), methylprednisolone only (2 of 22; 9%) and dexamethasone sodium phosphate (DSP) injectable and prednisone (4 of 22; 18.1%). Dosages of prednisone ranged from 0.214 to 2.12 mg/kg/day with an average of 1.06 mg/kg/day. Methylprednisolone doses ranged from 0.9 to 1.2 mg/kg/day with an average of 1.05 mg/kg/day. Of the four dogs receiving DSP, each received one subcutaneous injection followed by a maintenance dosage of prednisone (range 0.69–1.25 mg/kg/day; average 0.98 mg/kg/day). The average dose of DSP was 0.26 mg/ kg/day once with a range from 0.25 to 0.294 mg/kg. The median duration of corticosteroid therapy before onset of CC was 7.5 months (range 6–9 months).
这46只犬中有22只(47.8%)已知皮质类固醇的治疗剂量，而12只(26.0%)犬的治疗史或剂量未知，12只(26.0%)犬没有接受皮质类固醇治疗。诊断为CC的46只犬中有11只(23.9%)仅使用皮质类固醇治疗，10只(21.7%)使用硫唑嘌呤和皮质类固醇治疗，5只(10.8%)使用环孢素和皮质类固醇治疗，2只(4.3%)使用环磷酰胺和皮质类固醇治疗，1只(2.1%)使用环孢菌素、硫唑嘌呤、皮质类固醇和环磷酰胺治疗。5只犬(10%)在口服皮质类固醇的同时还服用了其他免疫抑制/免疫调节药物，包括长春新碱、来氟米特、四环素/烟酰胺、眼用环孢素和外用氢化可的松。由于使用激素治疗从而导致医源性糖皮质激素增多症的疾病如下：免疫性多发性关节炎(4/22；18.1%)、免疫介导性溶血性贫血(3/22；13.6%)、脊髓病(3/22；13.6%)、脑膜脑炎(3/22；13.6%)、过敏性皮肤病(2/22；9.1%)、落叶型天疱疮(2/22；9.1%)、免疫介导性血小板减少(2/22；9.1%)、再生障碍性贫血(1/22；4.5%)、疑似皮肤药物不良反应药疹(1/22；4.5%)；多灶性脑病(1/22；4.5%)。使用的皮质类固醇类型为单用泼尼松(16/22，72.7%)、单用甲基泼尼松龙(2/22，9%)和地塞米松磷酸钠(DSP)加泼尼松(4/22，18.1%)。泼尼松剂量0.214~2.12 mg/kg/d，平均1.06 mg/kg/d。甲基泼尼松龙剂量0.9~1.2 mg/kg/d，平均1.05 mg/kg/d。在接受DSP治疗的4只犬中，每只犬都接受了一次皮下注射，然后再接受泼尼松的维持量(范围0.69-1.25 mg/kg/d，平均0.98 mg/kg/d)。平均剂量为0.26 mg/kg/d，剂量范围为0.25~0.294 mg/kg。CC发病前激素治疗的中位持续时间为7.5个月(6-9个月)。
Histopathological findings
组织病理学检查
Thirty-seven of 46 (80%) of the histopathological samples were from antemortem excisional or punch biopsies, while nine of 46 (19.5%) were obtained at the time of postmortem examination.
46份组织病理学样本中有37份(80%)来自生前切除或打孔活检，而9份(19.5%)是在尸检时获得的。
A multinodular pattern of mineralization, including osseous metaplasia, was seen in 13 of 46 (28.2%) cases (Figure 1). A diffuse pattern was seen in 22 of 46 (47.8%; Figure 2), while a multifocal pattern was noted in 11 of 46 (23.9%; Figure 3).
46例中有13例(28.2%)呈多结节状矿化，包括骨化生(图1)。弥散性病变22例(47.8%；图2)，多灶性病变11例(23.9%；图3)。
Figure 1. Skin; canine, caliconsis cutis. Multinodular osseous metaplasia (arrow) with severe fibrosis in a dog with pituitary-dependent hyperadrenocorticism. Haematoxylin and eosin.
图1.皮肤；犬；皮肤钙化。患有垂体依赖性肾上腺皮质功能增多症的犬的多结节骨化生(箭头)伴严重纤维化。苏木精和伊红染料。
Figure 2. Skin; canine, caliconsis cutis. Diffuse mineral deposition extending from the superficial to mid-dermis. Haematoxylin and eosin.
图2.皮肤；犬；皮肤钙化。弥漫性矿物质沉积从真皮浅层延伸至真皮中部。苏木精和伊红染料。
Figure 3. Skin; canine, caliconsis cutis. Multifocal mineral deposition with significant inflammation and multinucleated giant cells (arrow) in a dog with iatrogenic hyperglucocorticism. Haematoxylin and eosin.
图3.皮肤；犬；皮肤钙化。医源性糖皮质激素增多症犬的多灶性矿物质沉积伴明显炎症和多核巨细胞(箭头)。苏木精和伊红染料。
Mineralization was present throughout all layers of the dermis in 27 of 46 (58.6%) cases, limited to the mid-dermis or deep dermis in six of 46 (13%) cases and extended from the superficial to mid-dermis in 12 of 46 (26%) cases. In four of 46 (8.6%) cases, mineralization was found in the subcutaneous tissue as well as the dermis, except for one dog where it was only in the subcutaneous tissue. Osseous metaplasia was observed in nine of 46 (19.5%) cases.
46例病例中，27例(58.6%)矿化累及真皮各层，6例(13%)局限于真皮中部或深层，12例(26%)于真皮浅至中层。46例中有4例(8.6%)在皮下组织和真皮中发现矿化，1例犬仅在皮下组织中发现矿化。46例病例中，有9例(19.5%)存在骨化生。
Epidermal atrophy was seen in 14 of 46 (30.4%) cases. Mild to moderate fibrosis was present in 29 of 46 (63%) cases, with scar tissue formation in nine of 46 (19.5%) cases (see Figure 1). Folliculitis and/or furunculosis were observed in 11 of 46 (23.9%) cases.
46例病例中有14例(30.4%)出现表皮萎缩。有29例(63%)出现轻至中度纤维化。有9例(19.5%)有瘢痕组织形成(见图1)。有11例(23.9%)出现毛囊炎和/或疖病。
Histological features in association with clinical risk factors
组织学特征与临床风险因素的关系
The incidence of histological features with each disease aetiology is summarized in Table 2. Due to confounding factors of multiple clinicians, dosages and diagnostic tests, and the difficulty of retrospectively assessing the clinical severity of the disease in individual dogs, a statistically supported correlation of disease aetiology and histological findings could not be made. Likewise, there was no evident correlation between breed with subjectively evaluated mineralization pattern, fibrosis and inflammation.
表2总结了每种疾病病因的组织学特征发生率。由于多个临床医生、剂量、诊断试验的混淆因素，以及很难在单个犬身上回顾评估疾病的临床严重性，所以无法在疾病病因学和组织学结果之间建立有统计学支持的相关性。同样，犬品种与主观评价的矿化模式、纤维化和炎症程度之间也没有明显的相关性。
Mid-dermal or deep dermal mineralization was seen in two dogs diagnosed with acute renal failure. Endogenous hyperadrenocorticism was diagnosed in seven of 14 (50%) dogs with epidermal atrophy evident on histopathology. Osteoma cutis was found in five of nine (55.5%) dogs with confirmed pituitary-dependent hyperadrenocorticism. No statistically significant correlations could be determined.
在两只诊断为急性肾衰竭的犬身上发现了真皮中部或真皮深部矿化。14只犬中有7只(50%)被诊断为内源性肾上腺皮质功能增多症，组织病理学显示表皮萎缩。9只确诊垂体依赖性肾上腺皮质增多症的犬中有5只(55.5%)出现皮下骨瘤。没有统计学上显著的相关性可以确定。
Mineral analysis
矿物质分析
Light microscopy revealed grains with very low birefringency (i.e. almost isotropic) among remnants of paraffin in three samples. The grains had a slightly higher refractive index than 1.4960. These optical characteristics are consistent with those of apatite [Ca5(PO4)3(OH,F,Cl)]. Similar grains were not observed in the fourth sample.
光学显微镜显示，在三个样品的石蜡残留物中，颗粒具有非常低的双折射(即几乎各向同性)。这些颗粒的折射率略高于1.4960。这些光学性质与磷灰石[Ca5(PO4)3(OH，F，Cl)]的光学性质一致。在第四个样品中没有观察到类似的颗粒。
The identification of those grains was undertaken by infrared spectrometry. Figure 4 compares infrared spectra of four samples with those of apatite and paraffin standards provided by commercial software installed in the spectrometer. Apatite has the primary and secondary peaks in wavenumber regions of 900–1200 and 500–700/ cm, respectively (see Figure 4b). The primary peak is characterized by a shoulder at the higher wavenumber side, and the secondary peak is a composite of two. As seen in Figure 4, the spectrum of the dog in Figure 4c showed two distinct peaks of apatite, suggestive of the highest content of apatite among the four samples examined. Apatite peaks were also observed for the dogs in Figure 4d and e, although they were less distinct than those for the dog in Figure 4c. However, unlike those three samples, the spectrum of the dog in Figure 4f did not show distinct apatite peaks, which was consistent with the light microscopic observations.
用红外光谱分析对这些颗粒进行了鉴定。图4将四个样品的红外光谱与安装在光谱仪中的商业软件提供的磷灰石和石蜡标准的红外光谱进行了比较。磷灰石的主峰和次峰分别位于900-1200和500-700/cm的波数区域(见图4b)。主峰的特征是在较高波数侧有一个肩峰，次峰是两个峰的组合。如图4所示，犬的频谱如图4c所示，存在两个明显的磷灰石峰，表明在四个样品中磷灰石的含量最高。图4d和e中的犬也观察到了磷灰石峰，但它们没有图4c中的犬那么明显。然而，与这三个样品不同的是，图4f中的犬的频谱没有显示出明显的磷灰石峰，这与光学显微镜观察一致。
Figure 4. Comparison of infrared spectra of four samples. (a) Paraffin (standard). (b) Apatite (standard). Three dogs reveal spectra consistent with apatite. (c) Dog with iatrogenic hyperglucocorticism secondary to treatment for insulinoma. (d) Dog with iatrogenic hyperglucocorticism secondary to treatment for multifocal brain disease. (e) Dog with pituitary-dependent hyperadrenocorticism. (f) Dog with unknown history.
图4. 比较了四例样品的红外光谱。(A)石蜡(标准)。(B)磷灰石(标准)。三例犬提示了与磷灰石一致的光谱。(C)继发于胰岛素瘤治疗的医源性糖皮质激素增多症的犬。(D)因治疗多灶性脑病而继发医源性糖皮质激素增多症的犬。(E)患有垂体依赖性肾上腺皮质功能增多症的犬。(F)病史不详的犬。
Discussion
讨论
The present study documents iatrogenic hyperglucocorticism as the major cause for CC in a cohort of 46 dogs, followed by endogenous hyperadrenocorticism and underlying renal disease. The underlying diseases were not known in four dogs. Male dogs were over-represented and large breeds are predisposed, with Pomeranians (2 of 46 dogs) representing the only small breed in this group. Rottweilers and Rottweiler/Labrador retriever mixes, Staffordshire terriers, boxers, boxer mixes, Akitas and Pomeranians are over-represented when compared with the hospital populations. Statistical analysis of the breed predisposition of CC has not been reported previously, except for documentation of increased risk for endogenous hyperadrenocorticism in boxers, Boston terriers, dachshunds and miniature poodles. While these were statistically significant findings, there were still small numbers of dogs with calcinosis cutis for each breed.
本研究记录了46例患有CC犬的主要病因是医源性糖皮质激素增多症，其次是内源性肾上腺皮质功能增多症和潜在的肾脏疾病。4例犬的潜在疾病尚不清楚。雄性犬的比例较高，且大型犬种更容易出现，博美犬(46例犬中的2例)是这一群体中唯一的小型犬种。与总病例数相比，罗威纳犬、罗威纳犬/拉布拉多寻回混血犬、斯塔福犬、拳师犬、拳师混血犬、秋田犬和博美犬的比例较高。除了在拳师犬、波士顿梗犬、腊肠犬和小型贵宾犬中内源性肾上腺皮质功能增多症的风险增加的文献外，以前还没有关于CC的品种易感性的统计分析。虽然这些都是统计上有意义的发现，但每个品种均仍有少量犬患有皮肤钙质沉积症。
The exact pathomechanism of CC is not clear. It is defined as deposition of insoluble, inorganic mineral salts in the dermis, epidermis or subcutis. The main factors necessary for tissue mineralization to occur include cation and anion deposition in tissues such as collagen or elastin. In normal skin, calcium and phosphate ions are in metastable equilibrium in the extracellular fluid. Changes in collagen fibrils may induce phase transformation of the ions, resulting in formation of crystal nuclei, which are deposited on organic matrices. Furthermore, with abnormally elevated mitochondrial calcium phosphate levels, crystal deposition and cell death may result.
CC的确切病理机制尚不清楚。它被定义为不溶性无机矿物盐沉积于真皮层、表皮或皮下组织中。组织矿化发生的主要必要因素包含阳离子和阴离子在胶原蛋白或弹性蛋白等组织中的沉积。在正常皮肤中，钙和磷酸盐离子在细胞外液中处于亚稳态平衡。胶原纤维的变化可能引起离子的相变，导致晶核的形成，并沉积在有机基质上。此外，线粒体磷酸钙水平异常升高，可能导致晶体沉积和细胞死亡。
The majority of the dogs included in this study developed CC due to iatrogenic hyperglucocorticism (54.3%) and endogenous hyperadrenocorticism (23.9%). The role of cortisol in the development of CC is considered a result of its gluconeogenic and protein catabolic activity. The rearrangement of the molecular structure of proteins leads to formation of an organic matrix that attracts and binds calcium. Furthermore, tissue damage may lead to dystrophic calcification and further increase of intracellular calcium influx. In humans, it is hypothesized that the ectopic soft tissue calcification in ‘idiopathic’ CC is due to increased amounts of c-carboxyglutamic acid, which contribute to the calcium and phospholipid binding.
本研究中纳入的大多数犬由于医源性糖皮质激素增多症(54.3%)和内源性肾上腺皮质激素增多症(23.9%)而发展为CC。在CC的发展形成中，皮质醇的作用被认为是糖异生和增加蛋白质分解代谢活性。蛋白质分子结构的重排形成能够吸引并结合钙的有机基质。此外，组织病变可能导致营养不良的钙化和细胞内钙内流的进一步增加。在人类中，特发性CC的异位软组织钙化被假定是由于促进钙和磷脂结合的γ-羧谷氨酸的增加。
Metastatic calcification occurs most commonly with chronic renal failure, as seen in six dogs (13.1%) in this study. Typically, the calcium-to-phosphorous (Ca:P) product exceeds 70 in order to result in metastatic calcification. Hypocalcaemia, with a resultant Ca:P product >70 was documented in only three of six dogs. There were insufficient numbers of serum biochemical profiles available to evaluate any further trends in the Ca:P product.
转移性钙化最常见于慢性肾衰竭，如本研究中的6只犬(13.1%)所示。通常情况下，钙磷比(Ca:P)超过70会导致转移性钙化。6只犬中的三只出现钙磷比大于70的低钙血症。没有足够数量的血清生化特征可用于评估Ca:P产品的进一步趋势。
Dogs with calcinosis cutis due to endogenous hyperadrenocorticism had a significantly higher median age when compared with dogs with iatrogenic hyperglucocorticism. The median age of dogs with endogenous hyperadrenocorticism was consistent with the previously reported age of onset for Cushing’s disease, as 9–11 years. Many of the dogs receiving corticosteroids had an underlying disease that may have originally occurred at a younger age. However, due to the small numbers of each disease entity, age of onset could not be correlated with a specific disease. Due to the small number of dogs with renal disease and the wide age distribution, there did not appear to be any correlation with age of onset of renal disease and onset of calcinosis cutis.
与患有医源性糖皮质激素过多症的犬相比，由于内源性肾上腺皮质机能增多症而患有皮肤钙质沉积症的犬的中位年龄明显更高。患有内源性肾上腺皮质功能增多症的犬的中位年龄与之前文献报道的库欣病发病年龄一致，为 9-11 岁。许多接受皮质类固醇治疗的犬有一种潜在的疾病，这种疾病最初可能发生在较年轻的时候。然而，由于每种疾病群体的数量很少，因此无法将发病年龄与特定疾病相关联。由于患肾病犬数量少，年龄分布广，肾病发病年龄与皮肤钙质沉积症发病年龄似乎没有任何相关性。
The distribution of skin lesions does not appear to be an indicator for a particular underlying cause for CC. In accordance with previous reports, the dogs had firm, palpable, erythematous papules to well-demarcated plaques, with multiple regions of the body affected. The subjective nature of the dermatological descriptions in the medical record precluded the ability to perform statistical analysis, but the dorsum appeared to be affected in all dogs with endogenous hyperadrenocorticism and was the most common body region affected with calcinosis cutis, while the inguinal and head lesions were the second most prevalent areas of calcinosis cutis (see Table 1).
皮肤病变的分布似乎不是衡量CC 特定潜在病因的指标。根据之前的报告，这些犬在机体多个区域有坚硬、可触及的红斑丘疹至边界清楚的斑块。病历中皮肤病学描述的主观性质无法进行统计分析，但在所有内源性肾上腺皮质功能增多症的病例中，犬的背部皮肤似乎都会患病，并且是最常见的皮肤钙质沉积的机体区域，而腹股沟和头部病变是皮肤钙质沉积症第二常见的区域(见表1)。
Inconsistency in documentation of medications administered did not allow documentation of a direct correlation of duration of administration of exogenous corticosteroids or other medication with the development of CC. The retrospective nature of the study precludes the ability to control for the exact dose of corticosteroids used in each dog, as well as to be able to account for any administration error or follow-up. Iatrogenic hyperglucocorticism with CC has been documented to develop after only 7 weeks of corticosteroid therapy or as late as 3 years of dexamethasone therapy every other day.
使用药物的记录不一致，导致无法将记录的外源性皮质类固醇或其他药物的治疗时间与CC的发展直接相关。该研究的回顾性性质使每只犬的皮质类固醇使用确切剂量不在控制中，包括误用药物和后续情况。有文献记载，CC的医源性糖皮质激素增多症仅在皮质类固醇治疗7周或每隔一天使用地塞米松治疗3年后发生。
No correlation was noted between the histopathological findings and underlying disease in dogs with CC. Neither pattern nor distribution of mineral deposits had distinct associations with underlying diseases or medication regimens. Moreover, the patterns and composition of inflammatory infiltrates, which were mostly composed of histiocytes and lymphocytes, as well as the amount of fibrosis, did not indicate particular underlying diseases.
在患有CC的犬中，组织病理学结果与潜在疾病之间没有相关性，矿物质沉积的模式和分布与潜在疾病或药物治疗方案也没有明显的关联。此外，炎性浸润的模式和组成(主要由组织细胞和淋巴细胞组成)以及纤维化的数量并未提示特定的基础疾病。
Epidermal atrophy, which is a cutaneous change often seen in dogs with hyperadrenocorticism, was seen in only 30.4% of the dogs in this study and included dogs with all patterns of mineralization. Although not as common as expected, this is probably due to the fact that inflammation elicited by mineral deposition resulted in epidermal hyperplasia. Subjectively, in most biopsies, the follicles appeared to be in telogen.
表皮萎缩是一种常见于肾上腺皮质功能增多症的犬的皮肤变化，在本研究包括所有矿化类型的犬中只有30.4%的犬出现。虽然不像预期的那么常见，但这可能是由于矿物沉积引起炎症导致了表皮增生。主观上，在大多数活检中，毛囊是处于休止期的。
Osteoma cutis was seen in the majority of dogs with pituitary-dependent hyperglucocorticism and a smaller percentage of dogs with iatrogenic hyperglucocorticism in this study, which has been recognized previously.
在这项研究中，皮下骨瘤出现在大多数患有垂体性糖皮质功能增多症的犬和一小部分患有医源性糖皮质激素增多症的犬上，这一点此前已经得到承认。
Polarizing microscopy and infrared spectrometry identified the deposited minerals as apatite in formalin-fixed tissues of three dogs with CC associated with different underlying diseases. The lack of identification of apatite in the fourth sample, from a dog with an unknown underlying disease, may be due to the small size of the available sample, because no mineral was found. In idiopathic CC in a human, the chemical composition of the mineral was determined qualitatively and quantitatively using both Fourier transform infrared and Raman microscopic techniques. The combination of techniques has revealed the most specific information on the composition of the deposits. The two major components in human idiopathic CC of subcutaneous tissues are type B carbonated apatite and β-carotene. In future studies, it may be prudent to perform Raman spectroscopy to determine whether β-carotene is also a component of CC in canine skin.
偏光显微镜和红外光谱分析表明，在福尔马林固定的三例与不同的潜在疾病有关的CC犬的组织中，沉积的矿物为磷灰石。在第四例样本中，没有发现磷灰石，可能是因为可用的样本太小，因为没有发现矿物。在人的特发性CC中，利用傅立叶变换红外法和拉曼显微技术定性和定量地测定了矿物的化学成分。技术的结合揭示了关于沉积物组成的最详细的信息。人类特发性皮下组织CC的两个主要成分是B型碳酸磷灰石和β-胡萝卜素。在未来的研究中，可能需要仔细考虑将拉曼光谱用来确定犬CC皮肤中是否存在β-胡萝卜素成分。
In conclusion, this study was unable to determine a correlation between underlying disease processes and the histopathological pattern of mineral deposition in the skin of dogs with CC. It was also evident that corticosteroid therapy plays a role in the development of CC, particularly in younger dogs, but it was uncertain whether dose ranges or a combination of medications influence the risk of mineral deposition. Certain breeds were over-represented that had not been previously documented. Owing to the confounding factor of multiple clinicians, dosages and diagnostic tests, and the difficulty of retrospectively assessing the clinical severity of the disease in individual dogs, a statistically supported comparison of disease aetiology and histological findings could not be made.
总之，本研究无法确定潜在疾病过程与CC犬皮肤矿物质沉积的组织病理学模式之间的相关性。很明显，特别是在年轻犬中，皮质类固醇的给药治疗在CC的发展中起作用，但剂量的范围或药物组合是否会影响矿物质沉积的风险并不确定。先前没有报道过某些品种的发病比例较高。由于多位临床医生、剂量和诊断试验的混淆因素，以及难以回顾性评估个体犬疾病的临床严重程度，无法对疾病病因学和组织学结果进行统计学比较。
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发表于 2023-9-22 15:44:09
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