Keratinocyte Biomarkers Distinguish Painful Diabetic Peripheral Neuropathy Patients and Correlate With Topical Lidocaine Responsiveness

Abstract

This study investigated quantifiable measures of cutaneous innervation and algesic keratinocyte biomarkers to determine correlations with clinical measures of patient pain perception, with the intent to better discriminate between diabetic patients with painful diabetic peripheral neuropathy (PDPN) compared to patients with low-pain diabetic peripheral neuropathy (lpDPN) or healthy control subjects. A secondary objective was to determine if topical treatment with a 5% lidocaine patch resulted in correlative changes among the quantifiable biomarkers and clinical measures of pain perception, indicative of potential PDPN pain relief. This open-label proof-of-principle clinical research study consisted of a pre-treatment skin biopsy, a 4-week topical 5% lidocaine patch treatment regimen for all patients and controls, and a post-treatment skin biopsy. Clinical measures of pain and functional interference were used to monitor patient symptoms and response for correlation with quantitative skin biopsy biomarkers of innervation (PGP9.5 and CGRP), and epidermal keratinocyte biomarkers (Nav1.6, Nav1.7, CGRP). Importantly, comparable significant losses of epidermal neural innervation (intraepidermal nerve fibers; IENF) and dermal innervation were observed among PDPN and lpDPN patients compared with control subjects, indicating that innervation loss alone may not be the driver of pain in diabetic neuropathy. In pre-treatment biopsies, keratinocyte Nav1.6, Nav1.7, and CGRP immunolabeling were all significantly increased among PDPN patients compared with control subjects. Importantly, no keratinocyte biomarkers were significantly increased among the lpDPN group compared with control. In post-treatment biopsies, the keratinocyte Nav1.6, Nav1.7, and CGRP immunolabeling intensities were no longer different between control, lpDPN, or PDPN cohorts, indicating that lidocaine treatment modified the PDPN-related keratinocyte increases. Analysis of the PDPN responder population demonstrated that increased pretreatment keratinocyte biomarker immunolabeling for Nav1.6, Nav1.7, and CGRP correlated with positive outcomes to topical lidocaine treatment. Epidermal keratinocytes modulate the signaling of IENF, and several analgesic and algesic signaling systems have been identified. These results further implicate epidermal signaling mechanisms as modulators of neuropathic pain conditions, highlight a novel potential mode of action for topical treatments, and demonstrate the utility of comprehensive skin biopsy evaluation to identify novel biomarkers in clinical pain studies.

Keywords: CGRP; IENF; keratinocytes; neuropathic pain; sodium channels.

Study Diagram

Visual outline of the study design and timeframe for subject participation.

Figure 1

Individual composite pain scores. A 4-week topical 5% lidocaine treatment resulted in a significant reduction in average pain scores for the PDPN and lpDPN cohorts. Pre-treatment (PRE) and post-treatment (POST) composite pain scores are shown for (A) the PDPN (Pain 1–Pain 21) and (B) the lpDPN (lp1-lp12) patient cohorts. Black stars represent the group average values with significant differences (p ≤ 0.05 by Wilcoxon-Mann-Whitney).

Figure 2

PDPN cohort individual clinical measures. Total PDPN cohort (n = 21) individual clinical symptoms responses (questionnaires, measures, composite sleep disturbance). Black stars represent the group average values with significant differences (p < 0.05 by paired Student's t-test). (A) Following a 4-week topical 5% lidocaine treatment, significant improvements were observed on the BPI-FPSI, but not the BPI-DLII. (B) Significant improvements were observed on the NPS (10) score. (C) No significant group changes were observed for the NIS-LL score, or (D) the NSS score. (E) Significant improvements were observed in group composite sleep disturbance scores.

Figure 3

PGP9.5+ cutaneous innervation. The panels depict representative images of PGP immunolabeling and DAPI fluorescence, PRE (pre-treatment period) and POST (post-treatment period) topical 5% lidocaine patch treatment, along with the half-sized grayscale PGP images used for counting, from a female and male PDPN patient compared with control. Thin white lines in the color images indicate the dermal/epidermal junction. Mag bar = 50 μM. Analysis of intraepidermal nerve fiber (IENF) measures (A) epidermal entry points and (B) epidermal endings, as well as (C) subepidermal axons and (D) upper dermal nerves, demonstrated significant losses across all cutaneous innervation sets from both PDPN and lpDPN cohorts compared with Control subjects. No innervation measures differentiated between the PDPN and lpDPN cohorts, and the 4-week topical 5% lidocaine treatment protocol had no significant effect on the innervation loss. Analysis of CGRP-positive (peptidergic) innervation revealed similar losses among the (E) subepidermal and (F) upper dermal compartments. CGRP-positive IENF were not identified among our human cohort biopsies (see results). *significant vs. PDPN and lpDPN; ^∧significant vs. PDPN.

Figure 4

Keratinocyte biomarker immunolabeling. Epidermal keratinocyte biomarkers are significantly increased among PDPN patients compared with control biopsies. The panels depict representative images of immunolabeling for (A) Nav1.6, (B) Nav1.7, and (C) CGRP along with DAPI fluorescence from PRE and POST treatment biopsies, along with half-sized grayscale images used for pixel intensity measures, from age-matched female and male PDPN patients compared to a male control. (D) There was a significant increase in PRE-treatment Nav1.6, Nav1.7, and CGRP immunolabeling among PDPN patients compared with control (brackets indicate significant differences). (E) Topical lidocaine treatment acted to decrease keratinocyte biomarker levels of Nav1.6, Nav1.7, and CGRP among PDPN patients such that no differences were found among the groups POST-treatment.