皮肤神经活体组织检查在周围神经病诊断中的应用

目的 探讨皮肤神经活体组织检查在周围神经病诊断中的作用,建立正常参考值范围,并比较临床表现、神经电生理检查与表皮神经纤维病变的一致性.方法 对51例有周围神经病症状和(或)体征的患者进行皮肤神经活体组织检查,计算表皮神经纤维密度(IENFD);同时收集10名健康志愿者作为对照.51例患者中,41例行常规肌电图及神经传导速度(NCV)检查.21例行皮肤交感反射(SSR),比较IENFD与NCV及SSR的一致性.结果 对照组与病例组相比,大腿IENFD(根/mm)分别为21.4±2.7及15.0±6.3(t=2.976,P=0.004);小腿IENFD分别为15.4±2.2及8.1±5.9(t=3.191,P=0.002).病例组与对照组相比大、小腿IENFD均有减少,差异有统计学意义.51例患者中,皮肤神经活体组织检查异常48例(94.1%),其中33例表现为长度依赖性周围神经病变;41例行常规肌电图检查,21例异常(51.2%);21例行SSR检查,异常17例(81.0%).仅表现为小纤维病变症状和(或)体征的29例患者中,27例(93.1%)皮肤神经活体组织检查异常;其中20例行NCV,异常6例(30.0%);14例行SSR,11例异常.结论 皮肤神经活体组织检查操作简单安全,对于以小神经纤维受累为主的周围神经病皮肤神经活体组织检查有较高的灵敏度.     

帕金森病患者皮肤小神经纤维病理研究

目的 探讨帕金森病（PD）患者皮肤小神经纤维的病理特点。方法 收集16例临床确诊并符合纳入和排除标准的PD患者和性别年龄匹配的16例健康对照者的临床资料和神经电生理结果,对PD症状及周围神经病变进行评估。所有受试者行小腿皮肤活组织检查,通过标准化泛轴突标记蛋白基因产物9.5(PGP9.5)和生长相关蛋白43(GAP-43)两种免疫组织化学染色定量表皮内神经纤维密度（Intraepidermal nerve fiber density,IENFD）,并进行统计学分析。结果 16例PD患者,男7例,女9例,年龄（66.000±9.675）岁,Hoehn and Yahr(H-Y)分期（1.906±0.612）。PD组尺神经、正中神经、腓肠神经远端感觉潜伏期较对照组延长（P<0.05）,余2组间神经电生理结果未见统计学差异（P>0.05）。PD组PGP9.5和GAP-43染色下IENFD值均低于健康对照组（8.618±3.984根/mm vs18.198±5.387根/mm,4.543±3.363根/mm vs 14.174±5.485根/mm;P<0.05）。两种染色下I...     

帕金森病患者皮肤神经磷酸化α-突触核蛋白研究

目的探讨帕金森病患者磷酸化α-突触核蛋白(phosphorylated α synuclein,p-α-syn)在皮肤神经中的沉积特点,以及其作为帕金森病外周生物标志物的可能性。方法纳入2017年6月1日至2018年8月31日就诊于郑州大学第一附属医院神经内科的15例帕金森病患者及同期年龄匹配的健康志愿者31名,进行13项小纤维神经病和症状问卷(SFN-SIQ)评分。将帕金森病患者按照主要临床表现分为运动迟缓(n=7)和静止性震颤(n=8)2个亚组,并以Hoehn-Yahr分级评价病情的严重程度,其中0~2.5级为早期组(n=11),3.0~5.0级为中晚期(n=4)。采用环形钻孔器取帕金森病患者小腿和颈部皮肤,健康对照组只取小腿部位皮肤,进行免疫组织化学和免疫荧光染色,观察p-α-syn在皮肤神经中的沉积情况并计数穿过单位长度基底膜的神经纤维数量即表皮内神经纤维密度(intraepidermal nerve fiber density,IENFD)。结果12/15的帕金森病患者表皮下神经丛、真皮神经束、汗腺、立毛肌、血管或毛囊周围神经中可见点状或线状p-α-syn沉积,健康对照组皮肤中未见沉积。p-α-syn在单个部位沉积的阳性率分别为小腿6/15、颈部7/15,总阳性率为12/15。帕金森病组的IENFD为(6.85±1.94)根/mm,较对照组[(10.45±3.70)根/mm]明显下降(t=-3.303,P=0.002),与SFN-SIQ评分呈负相关(r=-0.561,P=0.046)。疾病早期与中晚期患者之间,以及以震颤和以运动迟缓为主要表现的患者之间比较,p-α-syn沉积阳性率和IENFD差异均无统计学意义。结论p-α-syn在帕金森病患者皮肤神经纤维中沉积,伴随IENFD明显下降。提示皮肤神经中p-α-syn沉积可能是帕金森病固有的外周病理改变,有作为帕金森病患者诊断外周生物标志物的可行性。     

周围型神经纤维瘤病

神经纤维瘤病以典型的皮肤损害及明确的家族史,而易于诊断。本文介绍1例以具有诊断意义的皮肤病变,由于周围神经分布区有多数肿块,CT 扫描提示为神经纤维瘤病。病例:50岁女性,大腿后下部有一肿块,伴以钝痛。皮肤未见泸泡斑,即使回顾检查中也仅见左腹股沟有一孤立的牛奶咖啡色斑股部及小腿后面有感觉运动障碍,并可扪及多数无搏动的肿块,因而行 CT 检查.CT 显示沿腰神经丛分支分布区,以及右侧坐骨神经、股神经、腓总神经、胫后神经分布区可见多数软组织肿块。肿块中央密度低,CT 值为16Hu,数个肿块周围有钙化。根据 CT 检查提出多发性神经纤维瘤病的诊断。切除侵及右侧坐骨神经及其分支的肿块,证实为良性神经纤维瘤。审查其家庭,病人     

皮肤神经活检技术在诊断小纤维神经病中的运用

小纤维神经病(SFN)是临床常见的一种周围神经病,主要累及小直径的无髓及有髓纤维,临床主要表现为感觉症状和自主神经功能障碍,而无明显阳性体征。皮肤神经活检能显示躯体无髓神经纤维、有髓神经纤维及自主神经纤维,常用的方法有钻取活检及皮肤起疱法。通过明视野免疫组织化学染色和间接免疫荧光方法可计算表皮内神经纤维(IENF)密度。皮肤神经活检创伤小,能分别调查小纤维神经的不同功能,并有量化的观察指标,对SFN的诊断比腓肠神经活检更敏感,但此法无法确定各种周围神经疾病的原因。     

远端型遗传性运动神经病Ⅴ型一家系

目的 报道1个远端型遗传性运动神经病Ⅴ型(dHMN-Ⅴ)家系的临床、病理和基因改变特点.方法 该家系中连续4代有9例(4例男性,5例女性)在13～40岁之间发病,其中6例出现下肢无力;有2例女性患者仅出现双手肌肉无力和萎缩,其中1例出现锥体束征.先证者为20岁女性,13岁时发现双手肌肉萎缩和无力,15岁后出现下肢无力;肌电图提示神经源性损伤,神经传导速度检查显示运动神经复合肌肉动作电位波幅显著降低,伴随传导速度轻度减慢,感觉神经传导速度和动作电位波幅均正常.对先证者进行腓肠神经活体组织病理检查,并对先证者和其他4例发病者进行血Berardinelli-Seip先天性脂肪营养不良基因2(BSCL2)基因检查.结果 病理检查显示腓肠神经的有髓神经纤维数量轻度减少,伴随个别有髓神经纤维再生簇以及洋葱球样改变.基因检查显示BSCL2基因的第3号外显子存在263A→G杂合突变.结论 临床和基因检查证实该家系为dHMN-Ⅴ,其发病年龄和临床表现在同一家系中存在异质性,可以伴随轻微锥体束征和感觉神经损害.     

夏科-马里-图斯病2A2型一家系的临床病理特点

目的 报道一个早发型夏科-马里-图斯病(CMT)2A2家系,探讨其临床和病理特点.方法 该家系共有5例患者,呈常染色体显性遗传,先证者为36岁女性,6岁开始出现下肢进行性无力,8岁出现双足内翻.家族中另有2例男性和2例女性发病,发病年龄3～7岁,主要表现为缓慢进展的四肢远端肌肉无力、萎缩,伴随四肢远端感觉减退、腱反射减退及关节挛缩.先证者和其儿子的上肢感觉神经、下肢感觉和运动神经诱发电位波幅不能引出.对先证者左侧腓肠神经进行活体组织病理检查.对先证者和其他4例家系患者、3名无症状家系成员行MFN2基因测序.结果 病理检查可见腓肠神经有髓纤维数目重度减少,以大有髓神经纤维减少为主,伴随个别有髓神经纤维再生簇结构以及不典型的洋葱球样结构.电镜下可见轴索中线粒体聚集,未发现线粒体结构异常.5例患者存在MFN2基因R94W突变,无症状家系成员无此突变.结论 我国存在早发型CMT2A2家系,患者周围神经缺乏有髓神经纤维再生改变,提示MFN2基因突变对神经元的损害更大.     

诊断小神经纤维损伤的新靶点—皮肤活检

小神经纤维是一类直径小于7μm的小直径神经纤维,它们是周围神经纤维中最细小的部分,由于轴突直径和传导速率都非常小,与大直径神经纤维相比,更容易受到不利因素的损害,因此,小神经纤维受损症状常常是很多周嗣神经病的一个常见和早期的表现.但目前常规电生理等检查技术主要检测大直径有髓鞘神经纤维的功能,不能识别并检测广泛分布的小直径神经纤维,故小神经纤维损伤病变早期常被误诊为神经症、关节病等,因此,寻求一种能够检测小直径神经纤维的检查技术是目前临床亟待解决的问题.皮肤活检是一种能够对皮肤标本进行病理学观察的检查技术.它的应用为小神经纤维损伤的诊断提供了可能,并为周围神经病的早期诊断打开了新的窗口,现综述如下.     

经皮肤活体组织检查确诊的Lafora病五例

目的 探讨Lafora病的临床、病理特点及诊断方法.方法 总结分析5例经腋窝皮肤活体组织检查确诊的Lafora病患者的临床和组织病理学特点.5例患者都进行了腋窝皮肤活体组织检查,常规行HE、糖原(PAS)和阿辛蓝-糖原(AB-PAS)染色.结果 5例患者中4例青少年发病,1例成年起病.所有患者均表现为进行性加重的强直阵挛发作、肌阵挛和智能损害,可早期出现性格改变,构音障碍和行走不稳.腋窝皮肤活体组织检查发现Lafora包涵体存在于大、小汗腺的肌上皮细胞和导管细胞内.结论 经皮肤活体组织检查发现PAS阳性圆形或卵圆形包涵体,结合Lafora病典型的临床表现可以确诊该病;皮肤活体组织检查的部位可选择腋窝或腋窝以外的部位.     

增生期和成熟期皮肤瘢痕中的神经纤维形态学观察

摘要 背景 目前，虽有许多学者应用LSCM对神经轴突进行了研究，但对末梢神经的研究较少。目的 观察不同时期增生性瘢痕(HS)中神经纤维数量和形态的变化。 设计和时间 本实验为神经生理学对照实验，于2006年6月至2007年7月在山东大学附属省立医院进行。 实验对象 实验对象为创伤愈合后1个月至23年来院行瘢痕整复术的患者，共30例。 方法 标本取自烧伤或创伤后HS形成1月～23年的患者，分为增生期瘢痕组（≤6月）和成熟期瘢痕组(＞6月)，正常对照组取自患者的供皮区。利用单克隆抗神经丝蛋白(NF)抗体免疫荧光染色技术，在荧光显微镜和激光扫描共聚焦显微镜(LSCM)下观察不同时期瘢痕组织和正常皮肤组织中神经纤维的差异。 主要观察指标 本实验用神经丝蛋白免疫阳性神经纤维的阳性面积比作为评价神经支配强度的指标，同时对神经纤维形态学的改变进行重点描述。 结果 增生期瘢痕组神经纤维的阳性面积比明显高于正常对照组（P＜0.05），神经纤维分布杂乱，形态不规整，有局部的扭曲变细和肿胀断裂；成熟期瘢痕组神经纤维的阳性面积比明显低于正常对照组（P＜0.05），神经纤维排列较规整，内部结构和立体结构均接近正常。结论 瘢痕增生过程中神经纤维的密度和形态皆随时间变化而变化。     

Intraepidermal nerve fiber density of healthy human

Abstract

Objectives:

To analyze intraepidermal nerve fiber density (IEFND) by skin biopsy, evaluate the effect of age, anatomical sites, and ethnic origin on IEFND and develop a reference range of IENFD at the distal leg of healthy human.

Methods:

Seventy skin biopsy specimens from surgical procedures involving 70 patients were analyzed. Specimens were fixed routinely in formalin and thereafter embedded in paraffin. Nerve fibers of 10-μm-thick sections were observed using immunoperoxidase staining with panaxonal antibody protein gene product 9·5 (PGP 9·5). The morphology of intraepidermal nerve fibers (IENFs) and the IENFD was determined using light microscope. The statistical analysis was performed with SPSS 16·0 software.

Results:

No significant correlation was observed between IENFD and age (P_wrist = 0·830, P_{distal leg} = 0·478). The significant correlation was observed between IENFD and anatomic site (P = 0·001), the IENFD of upper arm and proximal thigh were significantly higher than that of wrist and distal leg. The reference range for IENFD of distal leg in normal Chinese humans was 40·55 fibers/mm². The IEFND of Chinese healthy human was significantly lower than that of Finnish (62·87 ± 15·25 vs 114·617 ± 32·322 fibers/mm², P < 0·05).

Discussion:

Skin biopsy may be a useful tool in sensory neuropathies. IENFD is independent of age, but varies in different parts of the body. The proximal sites have a higher IENFD, but no significant difference is found between the wrist and distal leg.     

Quantitative pathology of cutaneous nerve terminal degeneration in the human skin

Pathological diagnosis of neuropathy has traditionally depended on ultrastructural examinations of nerve biopsy specimens, particularly for sensory neuropathies affecting unmyelinated and small-myelinated nociceptive nerves. These sensory nerves terminate in the epidermis of the skin, and the pathology of neuropathy usually begins from nerve terminals. We investigated the feasibility of diagnosing small-fiber sensory neuropathy by evaluating cutaneous innervation. Skin biopsy specimens of 3-mm in diameter were obtained from the distal leg and the distal forearm of 55 healthy controls and 35 patients with sensory neuropathy. In the healthy controls, conventional intraepidermal nerve fiber densities (IENF densities) as measured using the image analysis system in the distal forearm and in the distal leg were correlated (r=0.55, P<0.0001), with significantly higher values in the distal forearm than in the distal leg (17.07+/-6.51 vs 12.92+/-5.33 fibers/mm, P<0.001). Compared to IENF densities of healthy controls, these values of neuropathic patients were significantly reduced in the distal forearm (5.82+/-6.50 fibers/mm, P<0.01) and in the distal leg (2.40+/-2.30, P<0.001). We further explored the possibility of quantifying skin innervation by counting "ocular intraepidermal nerve fiber density" (ocular nerve fiber density) with no aid of an image analysis system. This was based on the fact that the epidermal length on specifically defined sections was very close to the predicted epidermal length of 3 mm, the diameter of skin punches (P=0.14). Ocular nerve fiber densities were significantly correlated with IENF densities as measured by the image analysis system (r=0.99, P<0.0001). Dermal nerve fibers of neuropathic patients either disappeared or became degenerated. These findings were consistent with the notion of early terminal degeneration in neuropathy, and will facilitate quantitative interpretation of epidermal innervation in human neuropathy.     

Determinants of epidermal nerve fiber density in normal individuals

We studied the relationship between epidermal innervation and age, gender, height, and weight. Intraepidermal nerve fiber density (IENFD) of skin biopsies obtained from the proximal thigh and ankle of 84 normal individuals was quantified. A linear regression model was performed using IENFD at the thigh, IENFD at the ankle, and the thigh IENFD/ankle IENFD ratio, with age, gender, and height-weight interaction as predictors. An independent, negative correlation was found between age and IENFD at the ankle. No correlation was found between age and IENFD at the thigh. With increasing age the thigh IENFD/ankle IENFD ratio, a measure of the length-dependent distal-to-proximal gradient of epidermal nerve density, increased significantly. Gender, height, and body weight did not independently influence IENFD at either site. In normal individuals, distal epidermal innervation decreases in a length-dependent manner with advancing age. This must be considered when interpreting IENFD in disease states.     

Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy.

In previous work we demonstrated little effect of aging on the density and spatial pattern of epidermal innervation, however, this was restricted to two sites proximal and distal in the leg. To expand on these observations, we used punch skin biopsy in ten healthy controls to examine the variation in intra-epidermal nerve fiber (IENF) density at multiple specific sites in the leg. There was a consistent gradient in IENF from proximal to distal sites in all subjects, but minimal effect of age was noted. In the older age group (> or =70 years), the IENF densities ranged from 28.6+/-1.9 IENF/mm at the trunk to 15.5+/-1.5 at the distal leg. In a group of six patients with painful sensory neuropathy, we confirmed a length-dependent reduction in IENF. We observed what may be a predegenerative change, namely increased branching of epidermal nerve fibers at clinically unaffected sites. These data suggest little age-related change in IENF, at least up to age 75 years, in healthy normals. The increased branching complexity noted in unaffected sites in patients with sensory neuropathies implies that this may be a predegenerative change, preceding the actual loss of nerve fibers. Skin biopsy may be a useful tool for assessing the topographic extent and degree of nerve fiber damage in sensory neuropathies and its quantitative interpretation should be little affected by aging changes.     

Quantitation of sudomotor innervation in skin biopsies of patients with diabetic neuropathy

Previous assessments of the sudomotor system have depended on functional tests, and only a few studies document the pathologic findings of postganglionic nerve degeneration quantitatively and at the ultrastructural level. We developed a quantitative system of sudomotor innervation in skin biopsies of the distal leg by immunostaining of nerve fibers with anti-protein gene product 9.5 (PGP9.5) and by counterstaining with Congo red. A computerized area-based morphometric analysis was used to quantify the sweat gland innervation index (SGII), defined as the area of nerve fibers normalized to the area of sweat glands. This approach reduced the variations in measurements of sweat gland areas compared to the commonly used method by ～5.6-fold (2.47% ± 2.54% vs 13.97% ± 14.24%, p < 0.001); hence, variations in SGII were also reduced. We examined 35 Type 2 diabetic patients (24 men and 11 women; mean age, 56.5 ± 12.8 years), with symmetrical length-dependent neuropathy and reduced intraepidermal nerve fiber density (0.76 ± 0.95 fibers/mm). By light and electron microscopy, PGP9.5-positive nerve terminals surrounded Congo red-positive sweat gland secretory coils in controls; these periglandular nerve terminals were either absent or markedly reduced in diabetic patients. Diabetic patients had lower SGII values than age- and sex-matched controls (2.60% ± 1.96% vs 4.84% ± 1.51%, p < 0.0001). The SGII values were lower in patients with anhidrosis of the feet versus those with normal sweating of the feet (0.89% ± 0.71% vs 3.10% ± 1.94%, p < 0.01). Thus, skin biopsy offers combined assessment of sudomotor innervation.     

Epidermal nerve fiber density in sensory ganglionopathies: clinical and neurophysiologic correlations.

We assessed the involvement of somatic unmyelinated fibers in sensory ganglionopathies by skin biopsy and quantitative sensory testing (QST). Sixteen patients with ganglionopathy, 16 with axonal neuropathy, and 15 normal controls underwent skin biopsy at the proximal thigh and the distal leg. Intraepidermal nerve fibers (IENF) were immunostained by antiprotein gene product 9.5, and their linear density was quantified under light microscopy. Confocal microscopy studies with double staining of nerve fibers and basement membrane were also performed. Healthy subjects and neuropathy patients showed the typical proximodistal gradient of IENF density; in neuropathies, values were significantly lower at the distal site of the leg, confirming the length-dependent loss of cutaneous innervation. Conversely, ganglionopathy patients with hyperalgesic symptoms did not show any change of IENF density between the proximal thigh and the distal leg. The distinct pattern of epidermal denervation seen in sensory ganglionopathy reflected the degeneration of somatic unmyelinated fibers in a fashion that was not length-dependent, which was consistent with both clinical and neurophysiologic observations and supported the diagnosis.     

Skin biopsy and quantitative sensory testing do not predict response to lidocaine patch in painful neuropathies

Predictors of response to neuropathic pain treatment in patients with painful distal sensory neuropathies are lacking. The 5% lidocaine patch is believed to exert its effects on neuropathic pain via a local stabilizing effect on cutaneous sensory afferents. As such, it provides a model to assess whether the status of epidermal innervation as determined by skin biopsy or quantitative sensory testing (QST) of small- and large-diameter sensory afferents might serve as predictors of response to topical, locally active treatment. In this study we assessed associations between epidermal nerve fiber (ENF) densities, sensory nerve conduction studies (NCS), QST, and response to a 5% lidocaine patch in patients with painful distal sensory neuropathies. We observed no association between distal leg epidermal and subepidermal innervation and response to the lidocaine patch. Several patients with complete loss of distal leg ENF showed a response to the lidocaine patch. Similarly we observed no consistent association between treatment response and QST for vibration, cooling, warm, heat-pain, and cold-pain thresholds, or distal sensory NCS. Thus, distal-leg skin biopsy, QST, and sensory NCS cannot be used to identify patients with painful polyneuropathy likely to respond to a lidocaine patch in clinical practice. Further studies are required to clarify precisely the mechanism and site of action of the lidocaine patch in patients with peripheral neuropathic pain.     

Axonal fluorescence quantitation provides a new approach to assess cutaneous innervation

We present a novel approach to quantify skin innervation by measuring the PGP 9.5 immunoreactive (PGP-ir) fluorescence corresponding to axons within the epidermis and dermis. The skin biopsies from 35 controls and 45 small fiber neuropathy (SFN) patients were included. In 50-μm free-floating sections, we determined the intraepidermal nerve fiber density (IENFD) by direct fluorescence visualization and captured 2-μm thick individual optical sections using the same confocal microscope and magnification. We measured the fluorescence of the PGP-ir axons in both, epidermal and dermal area by using the ImageJ software. There was good interobserver and intraobserver reliability of PGP-ir measures, similar than for IENFD. The PGP-ir axons were found decreased in patients with SFN (1.1‰ and 9.0‰ respectively for epidermal and dermal area in contrast to 2.2‰ and 16.0‰, respectively to controls). The area under the ROC curve was 0.90 for the IENFD, 0.84 for epidermal PGP-ir axons and 0.70 for dermal PGP-ir axons. There was a positive correlation between the IENFD and the PGP-ir axons at epidermis (Spearman Rho=0.66, p<0.001) as well as for the dermal nerve length and the PGP-ir axons at dermis (Spearman Rho=0.45, p<0.05). This method is also particularly adequate for the quantitation of dermal nerve fibers. We conclude that quantifying the fluorescent PGP-ir axons could help to assess skin innervation (dermal and epidermal nerve fibers) in patients with SFN.