Figure 1.
The genotypic, anatomical and immunohistochemistry characterization of α-GalA KO mice.
The specific primers amplified bands of 295 bp for α-GalA(+/+) WT and 202 bp for α-GalA(−/−) and α-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; p = 0.0111). This trend was maintained and even increased after 12 week of age (n = 10 for WT, n = 5 for KO; p = 0.0023). 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.
Figure 2.
Mechanical and thermal sensitivity of α-GalA KO males.
Basal sensitivity towards mechanical stimulation (latency time (A); applied force (B)). Comparison of the basal sensitivity of male α-GalA KO (n = 16) and relative WT (n = 28) in response to mechanical stimulation, p<0.0001. Basal sensitivity to hot and cold temperature stimuli in male α-GalA KO (n = 34) and relative WT (n = 32), p<0.0001 as measured using the hot plate (C) and the acetone test (D) revealed the significant insensitivity of α-GalA KO (n = 19) males in comparison to their relative WT (n = 13), p = 0.0008. Basal insensitivity to noxious temperature of cold stimulus in male α-GalA KO (n = 7) and relative WT (n = 8), p = 0.0466 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), p = 0.0028 (F). Data are expressed as mean±SEM.
Figure 3.
Detection of ENFs in α-GalA KO males frontal paws.
The DAPI immunostaining of 50 µm floating sagittal mice frontal paw section with marked region of interest (A). The immunohistochemistry of α-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 α-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%; p = 0.0161) in α-GalA KO males in comparison to WT (C).
Figure 4.
The expression of pain receptors in α-GalA KO males frontal paws.
A) Immunohistochemistry of 12 µm frozen coronal sections of α-GalA KO males (n = 3) showed different expression at the protein level of the specific pain (Nav1.8), heat (TRPV1) and cold (TRPM8, Immunological Science) receptors in comparison to their WT controls (n = 3). B) The western blot analysis of Nav1.8 expression (∼220 kDa) in the whole protein extract from skin of frontal paws confirmed 1.5 fold increased protein expression of Nav1.8 in case of α-GalA KO males (n = 5) when compared to WT males (n = 5), p = 0.0246. The western blot analysis of TRPV1 expression (∼92 kDa) revealed 1.4 fold increased protein expression in case of α-GalA KO males (n = 3) when compared to WT (n = 3), p = 0.0344. The analysis of TRPM8 (Santa Cruz) expression (∼127 kDa) in the whole protein extract from skin of frontal paws showed the almost half expression decrease in protein expression of TRPM8 in case of α-GalA KO (n = 3) males when compared to WT (n = 3), p = 0.0518. Original images of presented blots are part of supplementary material (Figure S4). Scale bar represents 100 µm.
Figure 5.
The expression of Nav1.8 in epidermal neuronal fibers of α-GalA KO males frontal paws.
The evaluation of co-localization of Nav1.8 and PGP 9.5 expression in 50 µm floating sagittal sections of WT (A) and α-GalA KO males (B) (n = 3) revealed similar values of Pearson's coefficient in both cases (PWT = 0.16, PKO = 0.12). 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 α-GalA KO (D left panel, p = 0.1911). Panels C, D were enhanced with 1.125 zoom respectively to square area in figures A, B. Scale bars represent 100 µm.
Figure 6.
The expression of TRPV1 in epidermal neuronal fibers of α-GalA KO males frontal paws.
The evaluation of co-localization of TRPV1 and PGP 9.5 expression in 50 µm floating sagittal sections of WT (A) and α-GalA KO (B) males (n = 4) revealed similar values of Pearson's coefficient in both α-GalA WT males (PWT = 0.35) and KO (PKO = 0.29), (p = 0.1132). 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 α-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.