Can Wound Exudate from Venous Leg Ulcers Measure Wound Pain Status?: A Pilot Study

<div><p>We investigated the associations between the self-evaluated pain status and two pain biomarker candidates, nerve growth factor and S100A8/A9, in exudate from venous leg ulcer to finally develop an objective pain evaluation method. Patients with venous leg ulcer participated in this cross-sectional observational study conducted between April and October 2014 at two medical facilities. During routine wound care, each participant self-evaluated their pain status at each examination using the 10-point numerical rating scale (present pain intensity) and the short-form McGill Pain Questionnaire 2 (continuous pain, intermittent pain, neuropathic pain, affective descriptors, and total score). Venous leg ulcer exudate sample was collected after wound cleansing. The nerve growth factor and S100A8/A9 concentrations in the venous leg ulcer exudate were measured by enzyme-linked immunosorbent assay and standardized according to the wound area. The association between each pain status and the two standardized protein concentrations was evaluated using Spearman’s correlation coefficient. In 30 sample collected from 13 participants, the standardized nerve growth factor concentration was negatively correlated with continuous pain (<i>ρ</i> = -0.47, <i>P</i> = 0.01), intermittent pain (<i>ρ</i> = -0.48, <i>P</i> = 0.01), neuropathic pain (<i>ρ</i> = -0.51, <i>P</i> = 0.01), and total score (<i>ρ</i> = -0.46, <i>P</i> = 0.01). The standardized S100A8/A9 concentration was positively correlated with present pain intensity (<i>ρ</i> = 0.46, <i>P</i> = 0.03) and continuous pain (<i>ρ</i> = 0.48, <i>P</i> = 0.03). Thus, these two proteins may be useful for objective evaluation of wound pain in venous leg ulcer patients.</p></div

Participant characteristics (n = 13).

<p>Participant characteristics (n = 13).</p

Changes in continuous pain intensity and standardized protein concentrations in ID “G”.

<p>The protein concentrations were standardized according to the wound area, and then the standardized S100A8/A9 concentration was multiplied 10-fold for clarity. The horizontal axis indicates the duration in weeks from the first examination. Numbers of NGF data were 9, and of S100A8/A9 were 7 in spite of 10 time points because NGF was not detected in one sample from observation number 8, and S100A8/A9 was not detected in samples from observation numbers 10, 12, and 16. NGF = nerve growth factor.</p

Effects of AHL on wound healing and epithelialization in diabetic rats in experiment 2.

<p>(a) Macroscopic observation of healing of full-thickness wounds on both flanks of diabetic rats. The wounds on the right and left flanks were treated with vehicle (upper panels) or AHL (lower panels) on PWD 4. The appearances of both wounds before treatment on PWD 4 were similar. In AHL-treated wounds, by PWD 7, the necrotic tissue had disappeared and regeneration of the epidermis was apparent. Scale bar = 1 cm. (b) The wound area of AHL-treated wounds (red line) on PWD 8, 10 and 12 was significantly smaller than that of vehicle-treated wounds (blue line). *<i>P</i> < 0.05. (c) HE staining showed improvements of the histological abnormalities, including tylosis and invagination (arrow), in AHL-treated wounds but not in vehicle-treated wounds. Scale bar = 200 μm.</p

Immunohistochemistry for basement membrane components (a–f) and the proliferation marker Ki67 (g–i) in vehicle-treated (a, c, e and g) and AHL-treated wounds (b, d, f and i).

<p>Scale bar = 200 μm.</p

Associations between pain intensity and standardized NGF and S100A8/A9 concentrations.

<p>Associations between pain intensity and standardized NGF and S100A8/A9 concentrations.</p

Self-evaluated pain intensity (N = 30).

<p>Self-evaluated pain intensity (N = 30).</p

Wound and pain characteristics during each examination.

<p>Wound and pain characteristics during each examination.</p

Wound healing and epithelialization in nondiabetic and diabetic rats in experiment 1.

<p>(a) Macroscopic observation of the healing of full-thickness wounds on the right flanks of nondiabetic (upper panels) and diabetic rats (lower panels) until complete wound closure. Delayed formation of granulation tissue on PWD 4, abundant necrotic tissue on the wound bed on PWD 7 and 10, and delayed epithelialization on PWD 7 were evident in diabetic rats. Scale bar = 1 cm. (b) The wound area measured on PWD 7–9 was significantly greater in diabetic rats (red line) than in nondiabetic rats (blue line). *<i>P</i> < 0.05; **<i>P</i> < 0.01. (c) HE staining showed that the regenerating epidermis was shorter and thicker in diabetic rats than in nondiabetic rats. The histological abnormalities, including invagination of regenerating epidermis into the granulation tissue (arrow), were specific to diabetic rats. Scale bar = 200 μm.</p

<i>In vitro</i> analysis of the effects of AHL on keratinocytes.

<p>Foetal rat skin keratinocytes were treated with 10 μM AHL, vehicle only (0.1% DMSO), or were untreated (control). (a) Six hours after treatment, there were no morphological changes in any group. Scale bar = 200 μm. (b) Twenty-four hours after treatment, there were no differences in cellular proliferation among the three groups. (c) Expression analysis of genes related to epidermal basement membrane. (d) Qualitative analysis revealed significant increases in <i>Lm5</i> expression in AHL-treated cells compared with the vehicle-treated and control groups. Light gray: Control, dark gray: Vehicle, and black: AHL. **<i>P</i> < 0.01.</p