Skin Nerve Fibre Assay

PGPnerve

Skin Nerve Fibre Density

Since the epidermal nerve fibres were originally visualized by Paul Langerhans using the gold chloride staining, their assessment in skin seemed difficult and remained ambiguous for several decades. Only in the early 90's, the PGP9.5 pan-axonal marker was introduced, which confirmed the presence of nerve fibres in the skin. PGP9.5 has been used ever since to visualize aberrations of cutaneous neurites in different disease states and became a tool for the establishment of linear epidermal nerve fibre densities in the field of small fibre neuropathy. With the close control of the European Federation of Neurological Science (EFNS) and Peripheral Nerve Society (PNS), the PGP9.5 immunohistochemical technique has grown to a diagnostic test with level A recommendation. In direct answer and according to their guidelines and recommendations, we present an automated and thus standardized PGP9.5 immunofluorescence assay developed according to good clinical practice (GCP) guidelines. 

Results indicate that the automated PGP9.5 immunofluorescence assay reaches good agreement with the gold standard for skin biopsy immunostaining (EFNS) for linear nerve fibre densities in skin. The new technique shows a diagnostic performance characterized by an average sensitivity and specificity of 81.3% and 86.1 % with positive and negative predictive values of 72% and 91% respectively. Besides obtaining standardization and quality control at all levels of staining, this technique is easy to perform and does not require specialized fluorescence confocal microscopy. Though a quantitative caveat exists for the secondary branching in the epidermal nerve fibre network as assessed by the new technique, automated staining is proven to be adequate and reliable for diagnosing small fibre neuropathy and could be used as a first line testing tool. Strengthened by these observations and the good inter-observer agreement established, this new automated assay could easily become accessible for use in clinical trial and diagnostic testing.

Methods

As described in more detail by Ragé and colleagues (Ragé et al, 2010), paid healthy volunteers participated in an experimental capsaicin study, performed according to the methods used by Polydefkis (Polydefkis et al, 2004), which was approved by the Local Ethics Committee. The subjects received topical capsaicin application on three consecutive 24-hour cycles (72 hours) after which skin biopsies were assessed at variable time points. Biopsies assessed at day 1 (n=2), day 26 (n=1) and day 54 (n=2) were used. Five additional biopsy specimens, from diabetic subjects presenting poly-neuropathy, obtained from the study described by Ragé and coworkers (Ragé et al, 2011) were included in the study. Finally, 16 additional samples were obtained from healthy volunteers (15/16 from female subjects, age group 33-52 years) after written informed consent was obtained. 

Western Blot analysis was performed by SDS-page on cell lysates from A549 (Brichory et al, 2001; Tokumaru et al, 2008) and U87 (glioblastoma, Barrachina et al, 2007) cell lines exhibiting high PGP9.5 expression, to verify the accuracy of the rabbit polyclonal anti-human PGP9.5 antibody (RA95101, UltraClone Ltd.). 

After PGP9.5-immunofluorescence staining, image analysis was performed using the Axiovision Mozaik Imaging Software (Axiovision Rel 4.8) on an Axioplan 2 Imaging microscope equipped with motorized stage and z-stack features (Carl Zeiss, Germany). Virtual images were generated and used to perform all length measurements. The quantification of the linear density of nerve fibres was performed according to the EFNS guidelines (Lauria et al, 2005; England et al, 2009; Lauria et al (Joint task forces of EFNS and PNS), 2010) for all sections (figure 1).

NerveFibre2

Figure 1: Design of the Study: Skin biopsies were assessed at the lateral aspect of the distal leg and subsequently fixed in Zamboni's solution. After sucrose-cryoprotection and freezing, cryosections were made at 16µm for automated staining and 50µm for staining according to the gold standard. Stained tissue sections were digitalized using the Axipolan 2 Imaging microscope and Axiovision (Rel 4.8) software. Quality control (QC) was performed on each section with determination of correct staining in intrinsic controls (sweat glands, m.errector pili and/or nerve bundles) (scale bar – 20µm). The linear nerve fibre density was assessed by quantification according to EFNS-guidelines.

Results

Detailed illustration of method comparison will be described in a paper.

Brief Discussion

To provide in the need for standardization in diagnostic testing, we aimed to evaluate an accessible, reliable, first-line, automated staining method using PGP9.5-immunofluorescence. After the confirmation of good accuracy of the applied anti-human PGP9.5 rabbit polyclonal antibody using western blot analysis on A549 and U87 cell lysates and double labelling immunofluorescence with b-III-Tubulin in skin, the automated assay was challenged against the gold standard proposed by the EFNS (Lauria et al, 2005; Joint task force of the EFNS and PNS, Lauria et al, 2010). Although most investigators use bright field immunohistochemistry on serial 30-50µm-thick sections and normative values are only available for bright field (Lauria et al, 2010), the EFNS concluded that immunofluorescence results in a higher linear density of epidermal nerve fibers justifying our choice. Implementation of fluorescence in the diagnosis of SFN additionally opens the possibility of investigating multiple (nerve fiber) targets in a single tissue section. Full automation of the staining procedure could lead to accessible, stable and reliable testing in clinical trials and diagnostics. The automated PGP9.5 assay could serve as a tool in personalized medicine and could result in intra- and inter-laboratory concordance.

Publications in which HGX contributed

TEHPIFnew Myelin

Haploinsufficiency of VGluT1 but not VGluT2 impairs extinction of spatial preference and response suppression.
Callaerts-Vegh Z, Moechars D, Van Acker N, Daneels G, Goris I, Leo S, Naert A, Meert T, Balschun D, D'Hooge R.
Behav Brain Res. 2013 May 15;245:13-21. Epub 2013 Feb 5.
PMID:2339616

Mapping of carboxypeptidase m in normal human kidney and renal cell carcinoma: expression in tumor-associated neovasculature and macrophages.
Denis CJ, Van Acker N, De Schepper S, De Bie M, Andries L, Fransen E, Hendriks D, Kockx MM, Lambeir AM.
J. Histochem. Cytochem. 2013 Mar;61(3):218-35. Epub 2012 Nov 19.
PMID: 23172796

Asymptomatic small fiber neuropathy in diabetes mellitus: investigations with intraepidermal nerve fiber density, quantitative sensory testing and laser-evoked potentials.
Ragé M, Van Acker N, Knaapen MW, Timmers M, Streffer J, Hermans MP, Sindic C, Meert T, Plaghki L.
J Neurol. 2011 Oct; 258(10):1852-64. Epub 2011 Apr 7.
PMID: 21472496

The time course of CO2 laser-evoked responses and of skin nerve fibre markers after topical capsaicin in human volunteers.
Ragé M, Van Acker N, Facer P, Shenoy R, Knaapen MW, Timmers M, Streffer J, Anand P, Meert T, Plaghki L.
Clin. Neurophysiol. 2010 Aug; 121(8):1256-66. Epub 2010 Mar 26. PMID: 20347388

Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation and spatial reversal learning.
Balschun D, Moechars D, Callaerts-Vegh Z, Vermaercke B, Van Acker N, Andries L, D'Hooge R.
Cereb Cortex. 2010 Mar; 20(3):684-93. Epub 2009 Jul 2. PMID: 19574394

Contractile effects of 5-hydroxytryptamine (5-HT) in the equine jejunum circular muscle: functional and immunohistochemical identification of a 5-HT1A-like receptor.
Delesalle C, Van Acker N, Claes P, Deprez P, de Smet I, Dewulf J, Lefebvre RA.
Equine Vet J. 2008 Jun; 40(4):313-20. PMID: 18267888

IMPA1 is essential for embryonic development and lithium-like pilocarpine sensitivity.
Cryns K, Shamir A, Van Acker N, Levi I, Daneels G, Goris I, Bouwknecht JA, Andries L, Kass S, Agam G, Belmaker H, Bersudsky Y, Steckler T, Moechars D.
Neuropsychopharmacology. 2008 Feb; 33(3):674-84. Epub 2007 Apr 25.
PMID: 17460611

Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.
Martinet W, Abbeloos V, Van Acker N, De Meyer GR, Herman AG, Kockx MM.
J Pathol. 2004 Mar; 202(3):382-8. PMID: 14991905

Abstract March 2003

Calcification and gliosis in the hippocampus of adult rats with neonatal excitotoxic injury; S Nayak, D Ashton, M
Verhoye, N Van Acker and L Andries
International Congress on Schizophrenia Research, Colorado
JJPRD – Beerse, Bio-Imaging Lab - University of Antwerp, HistoGeneX – Antwerpen

Implementation of an automated pgp9.5 if assay to assess intra-epidermal nerve fiber density

Nathalie Van Acker1,6, Michael Ragé2, Ellen Sluydts1, Michiel W.M. Knaapen1, Martine De Bie1, Maarten Timmers4, Stefanie De Schepper1, Praveen Anand3, Theo Meert4, Léon Plaghki2, Patrick Cras5

1 HistoGeneX, Antwerp, Belgium
2 Institute of Neuroscience, Faculty of Medicine, Université catholique de Louvain, Brussels, Belgium.
3 Peripheral Neuropathy Unit, Hammersmith Hospital, London, UK
4 Janssen Research and Development, Janssen Pharmaceutica N.V. Beerse, Belgium.
5 Department of Neurology, Antwerp University Hospital, Born Bunge Institute, University of Antwerp, Belgium
6 Faculteit Geneeskunde en Gezondheidswetenschappen, University of Antwerp, Belgium