Nerve growth factor (NGF) is synthesized in cutaneous wound tissue, and its higher levels in the neonate may contribute to more efficient wound healing. We used in situ hybridization and immunohistochemistry to define NGF mRNA and protein expression in intact skin and following excision wounding in neonatal and adult rats. To determine whether NGF is associated with wound contractile fibroblasts (myofibroblasts), we also examined expression of alpha-smooth muscle actin (alpha-SMA) mRNA and protein, established markers for these cells. In intact skin, NGF mRNA and protein were present in vascular and arrector pili smooth muscle, hair follicle sheath cells, keratinocytes, and hypodermal fibroblasts. Neonatal adipocytes and Schwann cells also expressed NGF mRNA and protein, while adult adipocytes and Schwann cells displayed only NGF-ir. Following wounding, NGF mRNA expression was exuberant in these cell types, and increased similarly at both ages and appeared de novo in skeletal muscle cells. Additionally, both NGF mRNA and protein were present in macrophages and myofibroblasts, and expression in myofibroblasts was significantly greater in neonates. Wound myofibroblasts also expressed alpha-SMA. Surprisingly, after wounding alpha-SMA mRNA and protein were present in essentially all cells in which NGF mRNA was detected. We conclude that NGF expression is enhanced in many cell types after wounding, but greater NGF synthesis in neonates appears to be due to a more robust myofibroblast response. In addition, cell types which demonstrated NGF mRNA also expressed alpha-SMA, and staining for both markers increased following wounding, suggesting synthesis of both proteins is regulated in a coordinated fashion.

Contraction of skin excision wounds is affected by age and the presence of peripheral nerves. The present study examined relationships between peripheral innervation, wound contractile cells, and rate of wound closure to determine whether these are altered during development. Full-thickness 4-mm-diameter circular flaps were excised from the interscapular skin of rats on postnatal day (PND) 5, PND 12, or PND 60. Wounds of PND 5 and PND 12 rats contracted 45% between post-wound days (WD) 3 and 5 and more slowly thereafter, with a scar 9-14% of the original wound size by WD 21. In contrast, PND 60 wounds contracted only 22% between WD 3 and 5, and the residual scar at WD 21 was 40% of the original wound size. In younger rats, alpha-smooth muscle actin-immunoreactive myofibroblasts first appeared on WD 2 and attained maximum density at WD 5. Innervation, as assessed by protein gene product 9.5 immunoreactivity, appeared by WD 3 and increased rapidly through WD 7 in younger rats. In PND 60 wounds, myofibroblasts did not appear until WD 5 and did not attain a maximum until day 10. Nerve ingrowth was not significant until WD 10 and was depressed relative to younger rats throughout the healing phase. Wound nerves were predominantly immunoreactive to calcitonin gene-related peptide, and synaptophysin-immunostaining revealed close associations between varicosities and myofibroblasts. These findings suggest that wound myofibroblasts may be a target of peripheral nerves, and delayed wound closure in mature rats is associated with deficiencies in both myofibroblasts and innervation.

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Sympathetic axons are abundant within some orbital tissues but are absent from others. This study investigated cellular phenotypes of tissues containing sympathetic nerves en passage and compared these with phenotypes in regions devoid of sympathetic nerves and with smooth muscle targets. Two primary orbital smooth muscle targets, the tarsal muscle and orbital muscle, contained many synaptophysin-immunoreactive nerves. Target cells had ultrastructural features typical of smooth muscle and were immunoreactive for alpha-smooth muscle actin, smooth muscle myosin heavy chain, desmin, vinculin, and laminin, but not non-muscle myosin, vimentin, fibronectin, or type IV collagen; nerve growth factor (NGF) mRNA was detected by reverse transcription-polymerase chain reaction. Periorbital sheath devoid of sympathetic nerves contained elongated fibroblasts that were immunoreactive for vimentin, non-muscle myosin, and fibronectin, but not for alpha-smooth muscle actin, smooth muscle myosin heavy chain, vinculin, desmin, laminin, or type IV collagen, and did not express NGF mRNA. Regions of periorbital sheath containing sympathetic nerves had few synaptophysin-immunoreactive varicosities. Cells in this region contained myofilaments, ribosomes, and rough endoplasmic reticulum and were larger than tarsal muscle cells. They expressed NGF mRNA and showed a unique immunophenotype, reacting for vimentin, alpha-smooth muscle actin and myosin heavy chain, desmin, vinculin, laminin, and type IV collagen. This phenotype reflects both fibroblast and smooth muscle features similar to myofibroblasts or transdifferentiated smooth muscle described in other tissues. The spatial association between these cells and sympathetic nerves suggests that they may be involved in axon guidance or maintenance.