Modulatory Role of Sensory Innervation on Hair Follicle Stem Cell Progeny during Wound Healing of the Rat Skin

BACKGROUND: The bulge region of the hair follicle contains resident epithelial stem cells (SCs) that are activated and mobilized during hair growth and after epidermal wounding. However, little is known about the signals that modulate these processes. Clinical and experimental observations show that a reduced supply of sensory innervation is associated with delayed wound healing. Since axon terminals of sensory neurons are among the components of the bulge SC niche, we investigated whether these neurons are involved in the activation and mobilization of the hair stem cells during wound healing. METHODOLOGY/PRINCIPAL FINDINGS: We used neonatal capsaicin treatment to reduce sensory terminals in the rat skin and performed morphometric analyses using design-based stereological methods. Epithelial proliferation was analyzed by quantifying the number of bromodeoxyuridine-labeled (BrdU(+)) nuclei in the epidermis and hair follicles. After wounding, the epidermis of capsaicin-treated rats presented fewer BrdU(+) nuclei than in control rats. To assess SC progeny migration, we employed a double labeling protocol with iododeoxyuridine and chlorodeoxyuridine (IdU(+)/CldU(+)). The proportion of double-labeled cells was similar in the hair follicles of both groups at 32 h postwounding. IdU(+)/CldU(+) cell proportion increased in the epidermis of control rats and decreased in treated rats at 61 h postwounding. The epidermal volume immunostained for keratin 6 was greater in treated rats at 61 h. Confocal microscopy analysis revealed that substance P (SP) and calcitonin gene-related peptide (CGRP) receptor immunoreactivity were both present in CD34(+) and BrdU-retaining cells of the hair follicles. CONCLUSIONS/SIGNIFICANCE: Our results suggest that capsaicin denervation impairs SC progeny egress from the hair follicles, a circumstance associated with a greater epidermal activation. Altogether, these phenomena would explain the longer times for healing in denervated skin. Thus, sensory innervation may play a functional role in the modulation of hair SC physiology during wound healing

Distribution of the cholinergic nuclei in the brain of the weakly electric fish, Apteronotus leptorhynchus: Implications for sensory processing

Acetylcholine acts as a neurotransmitter/neuromodulator of many central nervous system processes such as learning and memory, attention, motor control, and sensory processing. The present study describes the spatial distribution of cholinergic neurons throughout the brain of the weakly electric fish, Apteronotus leptorhynchus, using in situ hybridization of choline acetyltransferase mRNA. Distinct groups of cholinergic cells were observed in the telencephalon, diencephalon, mesencephalon, and hindbrain. These included cholinergic cell groups typically identified in other vertebrate brains, for example, motor neurons. Using both in vitro and ex vivo neuronal tracing methods, we identified two new cholinergic connections leading to novel hypotheses on their functional significance. Projections to the nucleus praeeminentialis (nP) arise from isthmic nuclei, possibly including the nucleus lateralis valvulae (nLV) and the isthmic nucleus (nI). The nP is a central component of all electrosensory feedback pathways to the electrosensory lateral line lobe (ELL). We have previously shown that some neurons in nP, TS, and tectum express muscarinic receptors. We hypothesize that, based on nLV/nI cell responses in other teleosts and isthmic connectivity in A. leptorhynchus, the isthmic connections to nP, TS, and tectum modulate responses to electrosensory and/or visual motion and, in particular, to looming/receding stimuli. In addition, we found that the octavolateral efferent (OE) nucleus is the likely source of cholinergic fibers innervating the ELL. In other teleosts, OE inhibits octavolateral hair cells during locomotion. In gymnotiform fish, OE may also act on the first central processing stage and, we hypothesize, implement corollary discharge modulation of electrosensory processing during locomotion.Canada Foundation for Innovation http://dx.doi.org/10.13039/501100000196Canadian Institutes of Health Research http://dx.doi.org/10.13039/501100000024Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada http://dx.doi.org/10.13039/501100002790Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Le Fonds Québécois de la Recherche sur la Nature et les TechnologiesMerit Scholarship for Foreign StudentsPeer Reviewe

A mitochondrial-targeted antioxidant (MitoQ) improves motor coordination and reduces Purkinje cell death in a mouse model of ARSACS

Mitochondrial deficits have been observed in animal models of Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) and in patient-derived fibroblasts. We investigated whether mitochondrial function could be restored in Sacs−/− mice, a mouse model of ARSACS, using the mitochondrial-targeted antioxidant ubiquinone MitoQ. After 10weeks of chronic MitoQ administration in drinking water, we partially reversed motor coordination deficits in Sacs−/− mice but did not affect litter-matched wild-type control mice. MitoQ administration led to a restoration of superoxide dismutase 2 (SOD2) in cerebellar Purkinje cell somata without altering Purkinje cell firing deficits. Purkinje cells in anterior vermis of Sacs−/− mice normally undergo cell death in ARSACS; however, Purkinje cells numbers were elevated after chronic MitoQ treatment. Furthermore, Purkinje cell innervation of target neurons in the cerebellar nuclei of Sacs−/− mice was also partially restored with MitoQ treatment. Our data suggest that MitoQ is a potential therapeutic treatment for ARSACS and that it improves motor coordination via increasing cerebellar Purkinje cell mitochondria function and reducing Purkinje cell death

Keratin 6 expression in wounded epidermis.

<p>Back skin sections of control (A, C) and treated rats (B, D) were inmunostained for K6, a marker of epidermal activation. The edge of the wound is shown at the right of all the photomicrographs. At 32 h postwounding (A, B), the extension of K6 staining was equivalent in the epidermis of both groups. At 61 h (C, D) we found that the region of activated epidermis is expanded in capsaicin-treated rats when compared with control rats (arrowhead). epi, epidermis; der, dermis; sg, sebaceous gland; b, bulge; we, wound edge; hf, hair follicle. Scale bar = 200 µm.</p

The peptidergic fibers are more abundant in control rats during wound healing.

<p>The total length of nerve fibers immunoreactive for CGRP was estimated with the stereological probe of Space balls.</p

Epidermal proliferation of capsaicin-treated rats was reduced in the proximal region to wound edge.

<p>The total number of BrdU⁺ was estimated in the epidermis and hair follicles around the wound with the optical fractionator probe. For quantification we considered two regions: the inner region and the outer region from the wound edge. The total number of BrdU⁺ nuclei for the epidermal outer region is shown in A (exCtl, exCap) and for the inner region in B (inCtl, inCap). C and D show the number BrdU⁺ nuclei in the hair follicles in the outer and inner regions, respectively.</p

Bulge region of the rat hair follicle express neuropeptide receptors.

<p>Similar to mouse skin, label-retaining cells were found in the bulge region of rat hair follicles after 8 weeks of BrdU pulses. By confocal microscopy, we found that bulge region also display CD34 expression (A). Immunoreactivity for SP receptor (NK-1, <b>B</b>) and CGRP receptor (CLR, <b>C</b>) colocalized with CD34⁺ cells. Note that CLR immunoreactivity was especially intense in the region where CD34⁺ cells were located. The accessory protein RAMP-1, which determines CGRP binding, also colocalized with CD34 expression. Counterstaining was performed with propidium iodide (A3) and TOTO-3 (B3, C3, D3). Scale bar = 20 µm.</p

Epidermal activation is extended in capsaicin-treated rats.

<p>The graph shows the quantification of the volume of epidermis with K6 expression in control and treated rats. The volume was estimated by the stereological probe of Cavalieri. At 62 h, the volume of distal and proximal regions of epidermis with K6 immunoreactivity was significantly greater in treated rats than in control rats.</p

Sporadic cell death was detected in the hair follicles of capsaicin-treated rats.

<p>Epithelial apoptosis was evaluated by terminal dUTP nick end-labeling (TUNEL) assay in skin sections of control (A) and capsaicin-treated rats (B) at 51 h postwounding. Few stained nuclei for TUNEL (green arrows) were observed in the hair follicles of treated rats (B). Inset in B shows a higher magnification of a follicle with four labeled nuclei. Note that treated rats (B) presented regions of death epidermis (dotted line). Both groups presented labeled nuclei at the granulation tissue (C). A positive control was prepared by incubating skin sections with DNase I (D). epi, epidermis; der, dermis; sg, sebaceous gland; b, bulge; we, wound edge; hf, hair follicle. Scale bar = 200 µm; inset scale bar = 50 µm.</p

The migration of follicular cells toward the epidermis is altered in capsaicin-treated rats.

<p>The percentage of IdU⁺/CldU⁺ in the epidermis and hair follicles was determined at 32 h and 61 h postwounding from the total number of IdU⁺. The proportion of epidermal IdU⁺/CldU⁺ nuclei increased over time in the control group, whereas in the treated group decreased over time.</p