Biomedical subjects

N Stayner

Publications and source records attributed to N Stayner.

6 recordsLinked to original sources

PubMed1994

Localization of nerve growth factor (NGF) and low-affinity NGF receptors in touch domes and quantification of NGF mRNA in keratinocytes of adult rats.

Touch domes are clearly delineated mechanoreceptors that are visible on the depilated skin of mammals. These structures consist of a sharply circumscribed disk of thickened epithelium surmounting a group of Merkel cells that are innervated by type I sensory neurons. These characteristic cutaneous structures provide an ideal opportunity for investigating whether the localization of nerve growth factor (NGF) in the skin is related to sites of sensory axon termination. For these reasons, we have used immunocytochemistry to study the distribution of NGF and the low-affinity NGF receptor (p75NGFR) in the touch domes of adult rat skin. Intense NGF-like immunoreactivity was sharply restricted to keratinocytes (excluding the stratum corneum) of the thickened epidermis of touch domes. The epidermis immediately surrounding touch domes and the epidermis of the tylotrich hair follicle associated with touch domes were not stained by anti-NGF antiserum. Merkel cells of the basal epidermis of touch domes were immunonegative for NGF but were immunopositive for p75NGFR as were the type I nerve endings innervating these cells. Quantitative Northern blotting revealed that the level of NGF mRNA was substantially higher in keratinocytes isolated from the stratum granulosum and stratum spinosum than in keratinocytes isolated from the stratum germinativum. These findings indicate that NGF synthesis in mature skin has a highly restricted regional distribution that is primarily associated with the innervation of a specialized touch receptor.

Animals↗

PubMed1992

Serotonin-like immunoreactivity in Merkel cells and their afferent neurons in touch domes from the hairy skin of rats.

Immunoreactivity to serotonin was observed in Merkel cells as well as the afferent type I nerves terminating upon them in touch domes excised from the belly skin of rats. Type I nerves were strongly immunoreactive and could be traced through the dermis of the domal papilla. Merkel cell immunoreactivity was sometimes seen in the entire cell, but was often localized in the Merkel cell cytoplasm adjacent to nerve terminals and may have been in the terminals themselves. Domes were fixed by immersion in 4% paraformaldehyde-lysine-sodium-m-periodate (PLP) fixative at 4 degrees C for 2.5-3 hours and cryoprotected in 30% sucrose overnight. Sections were processed with the avidin-biotin complex peroxidase (ABC), peroxidase-antiperoxidase (PAP), and indirect immunofluorescence techniques with rabbit antiserum generated against serotonin.

Afferent Pathways↗

PubMed1992

Tactile function in skin-equivalent grafts.

Cultured grafts are excellent wound covers; however, their somatosensory capabilities are unknown. This is a preliminary report of a study which determined whether grafts of cultured skin become innervated and also examined whether seeding grafts with target tissue improved nerve growth or functional recovery. Autologous skin for grafting was generated from adult rat biopsy tissue. Dissociated keratinocytes were seeded on top of fibroblast-contracted collagen gels (skin-equivalents). Some animals received grafts composed entirely of skin-equivalents. Others had grafts with 2-mm punch biopsies (normal skin or touch domes) inserted into them. Prior to sacrifice, whole nerve recordings of the cutaneous nerves supplying the grafts were made following tactile mechanical stimulation of the graft surfaces. Tissue was processed for light and electron microscopy as well as silver stained. Nerve fibers were present in the dermis (generated from the fibroblast contracted collagen gels) of all animals and often extended to the epidermis. Light brushing of the cultured areas of the grafts produced little or no activity in the cutaneous nerves; however, afferent impulses were generated after rubbing the skin with a glass rod or pinching it with fine forceps. The implanted regions within the skin-equivalents varied from this pattern. Lightly brushing their surface resulted in vigorous activity in the nerves. Elements in the skin therefore seemed to enhance nerve regeneration and function. However, the quality of the engraftment was also important. Implanted regions of grafts experiencing poor "takes" had compromised innervation.

Animals↗

PubMed1992

Functional innervation of cultured skin grafts.

The aims of the present study were to determine 1) if grafts of cultured skin become innervated; and 2) whether tactile function of these grafts could be improved by implanting target tissue into them. Autologous skin equivalents were generated in vitro (30 d) for individual adult Sprague-Dawley rats. Some animals received pure skin equivalent grafts; others had target tissue consisting of 2-mm punch biopsies (normal skin or touch domes) inserted into their skin equivalents at the time of grafting. After 83 d, physiologic recordings were obtained from afferent nerves innervating the grafts. Tissue was processed for histology at various intervals. Silver staining of the tissues demonstrated many isolated nerve fibers in the dermis of cultured areas of skin as well as in implant zones. When grafts were rubbed with a glass rod or pinched with watchmaker forceps, impulses were evoked in nerves innervating both implant and cultured regions. In contrast, the afferent response to gently stroking grafts with a camel hair brush was severely reduced in cultured areas but was vigorous in implanted skin. Neuronal activity characteristic of type I neurons innervating touch domes was only found in cutaneous nerves innervating implants originally possessing domal tissue. Furthermore, grafts with good takes had better return of sensory function than grafts undergoing episodes of crusting. These results suggest that structural components or trophic factors present in implants enhanced the return of neural function related to the sensory modality of light touch; and this was also affected by the engraftment quality.

Action Potentials↗

PubMed1991

Skin wound closure in athymic mice with cultured human cells, biopolymers, and growth factors.

Skin wound closure remains a major problem in acute and reconstructive skin grafting after large burns because of limited availability of donor skin. This report evaluates six protocols for preparation in vitro of skin substitutes composed of cultured human cells, biopolymers, and growth factors for wound closure. Full-thickness wounds in athymic mice treated in a single procedure with cultured skin substitutes were compared directly to treatments with murine skin autograft, human skin xenograft, or no graft. Rectilinear planimetry of healed wounds 6 weeks after surgery showed that skin substitutes cultured in serum-free medium, and for 24 hours before surgery in defined medium with basic fibroblast growth factor (100 ng/ml), were not statistically different (p less than 0.05) in size from treatment with human skin xenograft. Acceptance and persistence of skin substitutes cultured in serum-free media were 70% at 6 weeks after surgery, as determined by staining of healed skin with a fluorescein-labeled monoclonal antibody against human HLA-ABC antigens. Ultrastructural examination of wounds with cultured human skin 6 weeks after treatment showed complete basement membrane, including anchoring fibrils, presence of melanocytes and pigment transfer to keratinocytes, and innervation of healed skin adjacent to basement membrane. These findings demonstrate effectiveness of cultured skin substitutes for closure of skin wounds and illustrate important capabilities to modulate the natural processes of wound repair, to increase supply of materials used for wound repair, and to enhance quality of wound healing.

Animals↗

PubMed1990

Primary culture of cells arising from a neuroendocrine skin carcinoma.

A carcinoma arising in the skin of the lip metastasized to the lymph nodes in the neck of a 53-year-old white man. Electron microscopy of the initial excisional biopsy specimen revealed that the tumor cells contained dense-cored vesicles (100 nm in diameter) in their cytoplasm and were joined by simple junctions. Cells from the nodal metastases were found to be immunoreactive for neuronspecific enolase, keratin intermediate filaments, and chromogranin A, but not for neurofilaments. The tumor was thus classified as a neuroendocrine skin carcinoma. In addition, its metastatic cells shared immunoreactive and ultrastructural characteristics of Merkel cells, which are situated in the basal epidermis of normal skin. Primary cultures from a nodal metastasis were established and characterized. The cells attached and proliferated on culture flask surfaces. The population-doubling time was 2 days. This is the first report where cells from a neuroendocrine skin carcinoma have been demonstrated to retain their characteristic ultrastructure in an in vitro environment (10 days). Studies of cells cultured from neuroendocrine skin carcinomas may prove useful in understanding the pathobiology of this disease and help define the in vitro growth requirements of nontransformed Merkel cells as well.

Carcinoma↗