Increased cutaneous NGF and CGRP-labelled trkA-positive intra-epidermal nerve fibres in rat diabetic skin

Neuroscience Letters 506 (2012) 59–63

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Increased cutaneous NGF and CGRP-labelled trkA-positive intra-epidermal nerve fibres in rat diabetic skin Laura Evans, David Andrew, Peter Robinson, Fiona Boissonade, Alison Loescher ∗ The Unit of Oral and Maxillofacial Medicine and Surgery, School of Clinical Dentistry, The University of Sheffield, Claremont Crescent, Sheffield S10 2TA, United Kingdom

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Article history: Received 4 May 2011 Received in revised form 14 October 2011 Accepted 19 October 2011 Keywords: Diabetes Neuropathic pain Nerve growth factor

a b s t r a c t In this study we have determined the amount of Nerve Growth Factor (NGF) and the innervation density of the glabrous hindpaw skin of diabetic rats (n = 4) and controls (n = 3). The proportion of intra-epidermal nerve fibres (IENF) expressing the high affinity NGF receptor (trkA) and calcitonin gene-related peptide (CGRP) were also determined. Four weeks after induction of diabetes by intraperitoneal streptozotocin injection skin was analyzed for: (i) NGF content using ELISA and (ii) the innervation density of peptidergic afferents that also expressed trkA using immunocytochemistry. NGF levels were approximately three-fold higher in diabetic skin compared to controls (diabetic: 134.7 ± 24.0 (SD) pg ml−1 , control: 42.7 ± 21.5 pg ml−1 , p = 0.002). As expected there was a significant reduction in IENF density in diabetic skin (2.7 ± 1.3 fibres mm−1 ) compared to controls (6.9 ± 1.5 fibres mm−1 ; p = 0.01). In diabetic rats there was no significant difference in the proportion of trkA-labelled IENF (diabetic 74 ± 21%; control 83 ± 15%, p = 0.6), but significantly more trkA-positive IENF were also labelled by CGRP antibodies in diabetic skin compared to controls (diabetic 89 ± 22%; control 38 ± 2%, p = 0.03). These data suggest that in diabetes the upregulation of cutaneous NGF may ‘over-troph’ the surviving axons, increasing CGRP labelling, which may be important in the aetiology of painful diabetic neuropathy. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Peripheral neuropathy is a frequent complication of diabetes, affecting about half of all patients [13]. In diabetic neuropathy (DN), long sensory nerve fibres (such as those innervating the lower limbs and the intercostal sensory fibres) degenerate, resulting in abnormal sensations that vary from mild tingling to severe pain. It has been estimated that painful DN affects up to 26% of diabetic patients [9]. Unfortunately, current medical treatments are largely inadequate as less than one third of all patients with painful-DN achieve 50% or greater pain relief [30]. In order to address this clinical need it is imperative that we understand more about the aetiology of painful DN to facilitate the development of mechanistic-based treatments. A recent study demonstrated that cutaneous nerve growth factor (NGF) is increased in the skin of mice with type II diabetes, and it was suggested that NGF might have an important role in the development of painful-DN [7]. NGF is known to be important in the development and maintenance of chronic pain [26,35], and intradermal injection of NGF evokes behavioural evidence of thermal and mechanical hyperalgesia in both animals and

∗ Corresponding author. Tel.: +44 114 2717849; fax: +44 114 2717863. E-mail address: a.loescher@Sheffield.ac.uk (A. Loescher). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.10.049

humans [1,4,21,25]. In the skin, NGF is produced by keratinocytes, macrophages and fibroblasts [12], where it can act on two different receptors: a low affinity receptor (p75) that is shared with other growth hormones, and a high affinity receptor (tropomyosinrelated kinase A, trkA), which facilitates the uptake of NGF in a subpopulation of sensory and sympathetic axons. In nociceptive fibres, NGF alters the expression of many ion channels [17,24] and neuropeptides [28,32], including calcitonin gene-related peptide (CGRP), a neuropeptide that has an important role in nociceptive signalling [33,34]. Mice that over-express cutaneous NGF have behavioural signs of thermal and mechanical hyperalgesia [10,29], as well as an increased density of CGRP-labelled nerve fibres [19]. In this study we have investigated if NGF is also upregulated in a rat model of type I diabetes, and whether there is an associated increase in the proportion of intraepidermal nerve fibres (IENF) that are immunopositive for trkA and CGRP (probable nociceptors).

2. Methods Tissue was obtained from four 11-week old male Sprague Dawley rats 4 weeks after induction of diabetes with 65 mg/kg streptozotocin (STZ) injected intra-peritoneally. All STZ-treated rats had blood glucose levels in excess of 20 mM at the time of tissue harvest. Age matched control rats (n = 3) had blood glucose levels lower than 10 mM at the time tissue harvest. Tissue was

L. Evans et al. / Neuroscience Letters 506 (2012) 59–63

obtained immediately after euthanasia by cervical dislocation. All the glabrous skin from both hindpaws was harvested immediately; tissue from the left hindpaw was frozen in liquid nitrogen then stored at −80 ◦ C for NGF quantification by ELISA, whereas tissue from the right hindpaw was immersion-fixed in 4% paraformaldehyde for immunohistochemistry. The lateral middle footpads taken from each hindpaw were analyzed with the investigator blind to the animal grouping. 2.1. ELISA measurements Samples were weighed and homogenised in ice-cold high-salt high-detergent extraction buffer containing protease inhibitors (Complete, Mini, EDTA-free Roche, UK). To solubilise and isolate NGF from binding proteins (including trkA) the samples were alkalised with 4 M NaOH to pH 11, cold centrifuged for 15 min, acidified with glacial acetic acid to pH 3, cold centrifuged for 30 min, neutralised with 4 M NaOH and cold centrifuged for 15 min [37]. The supernatant was assayed by colourimetry using a commercial kit (NGF Emax® ImmunoAssay System, Promega, UK) and the results read with an absorbance reader (Infinite M200, Tecan, Switzerland) at 450 nm. NGF content was quantified against a standard curve with known amounts of protein. Optical densities were adjusted by subtracting background, correcting for wet weight and dilution. Measurements were performed in duplicate and expressed as pg per mg of wet tissue weight per ml. The lower limit of sensitivity for the assay was 7.8 pg ml−1 of NGF. Statistical differences in the amount of NGF in diabetic and control tissue were determined with an unpaired Student’s t test. 2.2. Immunohistochemistry Tissue was fixed for 4 h in 4% paraformaldehyde and cryoprotected in 30% sucrose for 24 h. Tissue was then embedded longitudinally in Tissue-Tek O.C.T. (Optimal Cutting Temperature media; Sakura, Europe) and frozen at −80 ◦ C. Cryostat sections (10 ␮m) were collected onto poly-d-lysine (Sigma–Aldrich, UK) coated slides. Slides were washed in PBS 2 × 10 min and sections incubated with 10% normal donkey serum (NDS; Jackson ImmunoResearch Inc., USA) for 1 h in a moisture chamber at room temperature to reduce non-specific labelling. Primary antisera were diluted in PBS with 5% normal donkey serum and incubated at 4 ◦ C overnight. The antibodies used were anti-protein gene-product 9.5 raised in rabbit (PGP9.5; Ultraclone, UK diluted 1:1000); anti-trkA raised in goat (R&D Systems, UK diluted 1:5000) and anti-CGRP raised in guinea pig (Bachem, UK diluted 1:6000). After incubation in primary antisera the slides were washed 2 × 10 min in PBS, and then incubated in secondary antibodies that were conjugated to fluorophores and diluted in PBS with 1.5% normal donkey serum for 90 min at room temperature. PGP9.5 labelling was revealed with a donkey anti-rabbit antibody labelled with fluorescein isothiocyanate (FITC; Stratech UK, diluted 1:200); CGRP labelling was revealed with a donkey anti-guinea pig secondary antibody that was conjugated to indocarboyanine (Cy3; Stratech, diluted 1:500). TrkA labelling required signal amplification with tryamide conjugated to Coumarin (PerkinElmer Inc., USA) in order to increase the intensity of the weak labelling. Endogenous peroxidase activity was quenched after incubation with the primary antibody by washing the slides with PBS 3 × 6 min and then immersion in 0.3% H2 O2 and 0.1 sodium azide in PBS for 15 min. Sections were incubated in a donkey anti-goat secondary antibody that was conjugated to biotin (Stratech, diluted 1:5000). Horseradish peroxidase conjugated to streptavidin was used to deposit the coumarin-tyramide onto the tissue; the slides were then washed (2 × 10 min in PBS) and mounted in Vectorshield (Vector Laboratories) and coverslipped.

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