摘要
目的
1、建立皮肤神经活检的方法学,确定我实验室表皮神经纤维密度的正常参考值。
2、研究周围神经病变时皮肤神经病理的改变,比较表皮神经纤维密度与临床表现、神经电生理及腓肠神经活检结果的一致性。
3、研究糖尿病周围神经病时皮肤神经病理的改变及糖尿病周围神经病的相关危险因素。
4、研究痛性周围神经病变时皮肤神经病理的改变及探讨可能的发病机制。
方法
1、招募10名健康志愿者,分别取大小腿皮肤,进行蛋白基因产物9.5(protein gene product 9.5,PGP9.5)、血管活性肠肽(vasoactive intestinalpeptide,VIP)、P物质(substance P,SP)、降钙素基因相关肽(calcitonin-generelated protein,CGRP)的免疫组化染色,计算表皮神经纤维密度(intra-epidermalnerve fiber density,IENFD)的正常参考值,明确真皮神经丛及皮肤附属器的神经支配,确定VIP、SP、CGRP阳性纤维的分布和数量。
2、对65例周围神经病病人行皮肤神经活检,进行PGP9.5及各种神经肽的免疫组化染色,计算INEFD,并观察神经纤维形态。对每例病人进行详细的神经系统查体和感觉体征严重程度评分。其中57例患者做常规肌电图及神经传导速度(nerve conduction velocity,NCV)检查,40例做皮肤交感反射(skinsympathetic response,SSR)检查,18例行腓肠神经活检,对这些检查结果的一致性进行分析。
3、分析26例糖尿病周围神经病变患者皮肤神经病理改变的特点,探讨IENFD与病程及血糖、糖化血红蛋白的相关性。
4、对27例痛性周围神经病病人进行临床特点分析,比较疼痛及无痛患者IENFD及SP、CGRP阳性神经纤维数目的改变。
结果
1、用PGP9.5标记神经纤维,可见正常人表皮、真皮及皮肤附属器有丰富的神经纤维支配。我实验室IENDF正常值:大腿为21.44±2.73根/mm,小腿为15.44±2.20根/mm,与国外各实验室正常值相近似;大腿比小腿的表皮神经纤维密度约高40%。VIP阳性纤维表达于立毛肌、汗腺腺泡及导管;SP及CGRP阳性纤维分布于表皮及真皮层神经丛,神经纤细,数量少;CGRP阳性纤维多于SP阳性纤维。
2、周围神经病病人与健康志愿者相比,大小腿IENFD均有减少,差异有统计学意义。65例病人中,50例(76.9%)出现IENFD的异常,24例伴神经形态的改变;SCV异常者47.4%,SSR异常者62.5%。在小纤维神经病病人中,IENFD异常61.3%,SSR异常38.9%。在有大纤维受累的病人中,IENFD异常91.2%,SCV异常79.4%。小腿IENFD下降的严重程度与临床感觉症状评分相一致,与SCV异常相一致。腓肠神经总有髓纤维密度及大、小有髓纤维密度与小腿IENFD均高度相关(r=0.576、0.560、0.524,P=0.020、0.024、0.037)。
3、糖尿病病人小腿IENFD与病程负相关(r=-0.517,P=0.346),与血糖无相关性(r=-0.223,P=0.346),与糖化血红蛋白无相关性(r=-0.227,P=0.588)。糖耐量异常及糖尿病病人可在周围神经症状之前出现IENFD的下降。
4、小腿IENFD在疼痛组病人(6.54±6.51根/mm)及无疼痛组病人(8.54±6.09根/mm)差异无显著性意义(P＞0.05):SP阳性纤维在疼痛组(2.18±2.30根/4.5mm)较无疼痛组(5.23±4.00根/4.5mm)显著减低(P＜0.05):CGRP阳性纤维在疼痛组病人(12.56±8.48根/mm)较无疼痛组(21.04±10.33根/mm)显著减低(P＜0.05)。
结论
1、皮肤神经活检简单安全,PGP9.5免疫组化染色计算IENFD方法可靠。SP、CGRP及VIP阳性纤维有不同的分布,可用于不同类型神经纤维的鉴别。
2、皮肤神经病理检查可用于小纤维神经病的早期诊断,可以发现无症状的周围神经病患者。除可出现IENFD的改变外,神经纤维形态的改变也可提供诊断价值。皮肤神经活检可以观察远近端神经支配的不同。IENFD下降程度与SCV异常及有髓神经纤维密度减低相一致,对于感觉为主的周围神经病,皮肤神经活检较腓肠神经活检更敏感。
3、糖尿病周围神经病患者IENFD下降,即使无症状患者也可以有IENFD的下降,提示皮肤神经活检可以发现早期临床下的糖尿病周围神经病。随糖尿病病程的延长,表皮神经纤维脱失程度逐渐加重。
4、有疼痛组病人SP、CGRP阳性纤维较无疼痛组显著降低,提示SP、CGRP可能与疼痛的发生有关。
Objective
1. To develop a normative reference values of epidermal nerve fiber density in healthy control subjects. To mark different fiber types using different peptides.
2. To compare the concordance between clinical features, electrophysiology and the results of skin biopsy results in the patients with peripheral neuropathies.
3. To investigate skin nerve changes in diabetic neuropathy. To evaluate correlation between IENFD and duration of diabetes, glucose, glycosylated hemoglobin.
4. To investigate skin nerve changes in painful peripheral neuropathy. To discuss possible mechanism of pain.
Methods
1. 10 volunteers (age range, 21-43years )were examined. Each subject had 2 punch biopsies performed in the thigh and distal part of the leg. Immunocytochemistry for the general neuronal marker protein gene product 9. 5( PGP9. 5) and three neuropeptides (Calcitonin gene-related peptide CGRP, subsantce P SP, Vasoactive intestinal polypeptide VIP)were performed on 10 volunteers.
2 .We studied 65 patients with peripheral neuropathies . Skin biopsies were obtained from distal leg and/or proximal leg and nerve identified using immunohistochemistry with antibody to protein gene product (PGP)9.5 and neuropeptides. Detailed physical examination were performed in each patient. 57/65 performed routine nerve conduction velocity and electromyography, 40/65 performed SSR, 18/65 performed sural nerve biopsy. The concordance of the consequences was compared. 3. Analyse changes of skin nerves in 26 cases of diabetic neuropathy. Discuss correlation between IENDF and duration of diabetes , as well as glucose and glycosylated hemoglobin.
4. Analyse changes of skin nerves in 27 cases of painful peripheral neuropathy. Discuss correlation between symptoms and immunocytochemistry of PGP9. 5 and neuropeptides .
Results
1.The distribution of PGP9.5 immunoreactive nerve fibers were seen abundantly in the epidermis, dermis, around sweat glands, arrectores pilorum and hair follicles. We enumerated intra-epidermal nerve fibers per millimeter to derive a linear density (Intraepidermal nerve fiber density , IENFD). IENFD was 21. 44±2.73 IENF/mm (mean±SD) in thigh and 15.44±2. 20IENF/mm(mean±SD) in the distal part of the leg. VIP immunoreactive nerve was seen in autonomic nerve fibers around sweat glands and arrectores pilorum . CGRP immunoreactive nerve was found in a population of sensory nerve fibers and around sweat glands. SP immunoreactive nerve was seen in sensory nerve, which was spare compared to CGRP immunoreactive nerve.
2. The intraepidermal nerve fiber density were significant lower in patients than in healthy controls both in proximal and distal legs. 50/65(76.9%) patients showed abnormalities in skin biopsy, in which 24 patients accompaniment with morphological changes of skin nerves, length-dependent neuropathy. In the whole cohort , 57 have performed route electrophysiology, in which 27(47.4%) were abnormal. 40 have performed SSR, in which 25(62. 5%) were abnormal. IENFD correlated with the densities of sural nerve total myelinated fibers(r=0.576 , P=0.020), small myelinated fibers(r=0.524, P=0.037), and large myelinated fibers(r=0.560, P=0.024)
3. The intraepidermal nerve fiber density were significant lower in patients with diabetes. There were significant inverse correlations between IENFD and the duration of diabetes. There was no significant difference between IENFD and glucose, glycosylated hemoglobin.
4. There was no significant difference of IENFD between patients with and without pain. SP- and CGRP- positive fibers were significant lower in patients with pain than those without pain.
Conclusion
1.Skin biopsy was safe and tolerated. It was useful to assess the distribution of small fibers in skin. Skin biopsy may have a role in the assessment of small fiber neuropathy.
2. Skin biopsy can play an important role in the diagnosis of peripheral disorders. Besides reduced IENFD .morphological changes in skin nerve fibers might be an early sign of peripheral neuropathy. IENFD correlated with the densities of sural nerve total myelinated, small myelinated and large myelinated fibers. Distal leg IENFD may be more sensitive than sural nerve biopsy in small fiber sensory neuropathy.
3. In patients with diabetic neuropathy , skin biopsy can reveal loss of small-diameters nerve fibres even there were no symptoms. The extent of epidermal denervation rises with the duration of diabetes.
4. In patients with painful symptoms , skin biopsy can reveal loss of SP-and CGRP- positive fibers more severe than those who have no pain.
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