The expression of TRPV1 in epidermal neuronal fibers of <i>α</i>-GalA KO males frontal paws.

<p>The evaluation of co-localization of TRPV1 and PGP 9.5 expression in 50 µm floating sagittal sections of WT (A) and <i>α</i>-GalA KO (B) males (n = 4) revealed similar values of Pearson's coefficient in both <i>α</i>-GalA WT males (<i>P_WT = 0.35</i>) and KO (<i>P_KO = 0.29</i>), (<i>p = 0.1132</i>). The TRPV1 receptor is expressed in neuronal fibers of WT (C left panel) males marked by specific antibody PGP 9.5 with the high intensity as it is expressed in <i>α</i>-GalA KO (D left panel). The co-localization of both markers (C, D right panels) revealed similar results. Panels C, D were enhanced with 1.125 zoom respectively to square area in panels A, B. Scale bars represent 100 µm.</p

The expression of Nav1.8 in epidermal neuronal fibers of <i>α</i>-GalA KO males frontal paws.

<p>The evaluation of co-localization of Nav1.8 and PGP 9.5 expression in 50 µm floating sagittal sections of WT (A) and <i>α</i>-GalA KO males (B) (n = 3) revealed similar values of Pearson's coefficient in both cases (<i>P_WT = 0.16</i>, <i>P_KO = 0.12</i>). The neuropathic pain receptor Nav1.8 is expressed in neuronal fibers of WT (C left panel) males marked by specific antibody PGP 9.5 with the same intensity as it is expressed in <i>α</i>-GalA KO (D left panel, <i>p = 0.1911</i>). Panels C, D were enhanced with 1.125 zoom respectively to square area in figures A, B. Scale bars represent 100 µm.</p

Pain Related Channels Are Differentially Expressed in Neuronal and Non-Neuronal Cells of Glabrous Skin of Fabry Knockout Male Mice

<div><p>Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (<i>α</i>-GalA) enzyme. The <i>α</i>-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the <i>α</i>-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The <i>α</i>-GalA null mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the <i>α</i>-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD.</p></div

The genotypic, anatomical and immunohistochemistry characterization of <i>α</i>-GalA KO mice.

<p>The specific primers amplified bands of 295 bp for <i>α</i>-GalA(+/+) WT and 202 bp for <i>α</i>-GalA(−/−) and <i>α</i>-GalA(−/0) KO. In case of founders females the heterozygosity was confirmed by PCR amplification of both WT and KO bands (A). The body weight of Fabry males was significantly increased just after 8 weeks (n = 15 for WT, n = 20 for KO; <i>p = 0.0111</i>). This trend was maintained and even increased after 12 week of age (n = 10 for WT, n = 5 for KO; <i>p = 0.0023</i>). Data are presented as fold of body weight increase in KO males related to the mean of WT males (B). Hematoxylin-eosin staining of 12 µm frozen frontal paws sections (C). Immunohistochemistry of frontal paw sections clearly shows the accumulation of globotriaosylceramide (Gb3; red) in the epidermis and stratum corneum of KO in comparison to WT mice (D). Scale bar represents 100 µm. Graphical data are expressed as mean±SEM.</p

Mechanical and thermal sensitivity of <i>α</i>-GalA KO males.

<p>Basal sensitivity towards mechanical stimulation (latency time (A); applied force (B)). Comparison of the basal sensitivity of male <i>α</i>-GalA KO (n = 16) and relative WT (n = 28) in response to mechanical stimulation, <i>p<0.0001</i>. Basal sensitivity to hot and cold temperature stimuli in male <i>α</i>-GalA KO (n = 34) and relative WT (n = 32), <i>p<0.0001</i> as measured using the hot plate (C) and the acetone test (D) revealed the significant insensitivity of <i>α</i>-GalA KO (n = 19) males in comparison to their relative WT (n = 13), <i>p = 0.0008</i>. Basal insensitivity to noxious temperature of cold stimulus in male <i>α</i>-GalA KO (n = 7) and relative WT (n = 8), <i>p = 0.0466</i> as measured via cold plate assay (E). The data from plantar cold sensitivity assay confirmed the observed insensitivity of KO males (n = 10) to cold stimuli when compared to WT males (n = 10), <i>p = 0.0028</i> (F). Data are expressed as mean±SEM.</p

Detection of ENFs in <i>α</i>-GalA KO males frontal paws.

<p>The DAPI immunostaining of 50 µm floating sagittal mice frontal paw section with marked region of interest (A). The immunohistochemistry of <i>α</i>-GalA KO males (n = 3) revealed the scattered expression of PGP9.5 (red) - specific marker of neuronal terminations in the epidermis of frontal paw skin in comparison to their WT controls (n = 3). The dermis and epidermis border was distinguished by staining for Collagen IV (green) and visually determined by dotted lines. Paw epidermal PGP9.5 positive fibers showed morphological abnormality such as fragmentation in <i>α</i>-GalA KO males, whereas the epidermal fibers showed a more regular morphology in WT males (white arrows) (B). Scale bar represents 100 µm. Numerical analysis of neuronal fibers terminations showed significant decrease (about 50%; <i>p = 0.0161</i>) in <i>α</i>-GalA KO males in comparison to WT (C).</p

Skin Gb3 deposits in FD patients with classical GLA mutation.

<p>Confocal microscope (x40 in A and B; x60 in C) study of Gb3 in skin vessels (A, AI and AII), sweat gland tubules (B, BI and BII) and arrector pilorum muscle (C, CI and CII). A) Skin arterioles showed no Gb3 deposits in the control subject (A). However, these abnormal deposits were abundant in the male FD patient in the wall of the arteriole and its endothelium (AII) and less evident in the female patient (AI); B) Sweat gland tubules of the control showed no Gb3 deposits (B), which were found in the male with FD (BII). The female showed nearly absent Gb3 deposits inside the sweat gland tubules (BI); C) Arrector pilorum muscle cells of the control have no Gb3 deposits (C) but they were found in the male (CII) and less in female FD patient (CI).</p

Clinical and laboratory findings in patients with Fabry disease.

<p>Clinical and laboratory findings in patients with Fabry disease.</p

Epidermal nociceptive innervation in FD patients with classical GLA mutation and a control subject.

<p>Leg epidermal innervation disclosed by confocal microscope (x40) in an age-matched control subject (A), female (B) and male (C) FD patients. Nerve fibres in red and collagen staining in green. Free-ending PGP immunoreactive nociceptive fibres crossing the dermal–epidermal junction marked by collagen (arrow) are evident in the epidermis of the control (A). FD patients showed a decrease in these fibres, which mainly characterized the male (C) and to a lesser extent the female (B) patient.</p

Comparison between patients with classical (skin Gb3 positive) and non-classical (Gb3 negative) GLA mutations.

<p>Comparison between patients with classical (skin Gb3 positive) and non-classical (Gb3 negative) GLA mutations.</p