Spinal cord from body donors is suitable for multicolor immunofluorescence.

Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level. Obtaining fresh neuronal tissue from human organ donors is difficult and sometimes impossible. In anatomical body donations, neuronal tissue is dedicated to research purposes and because of its easier availability, it may be an alternative source for research. In this study, we harvested spinal cord from a single organ donor 2 h (h) postmortem and spinal cord from body donors 24, 48, and 72 h postmortem and tested how long after death, valid multi-color immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry is possible. We used general and specific neuronal markers and glial markers for immunolabeling experiments. Here we showed that it is possible to visualize molecularly different neuronal elements with high precision in the body donor spinal cord 24 h postmortem and the quality of the image data was comparable to those from the fresh organ donor spinal cord. High-contrast multicolor images of the 24-h spinal cords allowed accurate automated quantification of different neuronal elements in the same sample. Although there was antibody-specific signal reduction over postmortem intervals, the signal quality for most antibodies was acceptable at 48 h but no longer at 72 h postmortem. In conclusion, our study has defined a postmortem time window of more than 24 h during which valid immunohistochemical information can be obtained from the body donor spinal cord. Due to the easier availability, neuronal tissue from body donors is an alternative source for basic and clinical research.

Structural and molecular characteristics of axons in the long head of the biceps tendon.

The innervation of the long head of the biceps tendon (LHBT) is not sufficiently documented. This is a drawback since pathologies of the LHBT are a major source of shoulder pain. Thus, the study aimed to characterize structurally and molecularly nervous elements of the LHBT. The proximal part of 11 LHBTs was harvested intraoperatively. There were 8 female and 3 male specimens. Age ranged from 66 to 86 years. For structural analyses, nervous elements were viewed in the transmission electron microscope. For molecular characterization, we used general neuronal markers including antibodies against neurofilament and protein gene product 9.5 (PGP9.5) as well as specific neuronal markers including antibodies against myelin basic protein (MBP), calcitonin gene-related product (CGRP), substance P (SP), tyrosine hydroxylase (TH), and growth-associated protein 43 (GAP43). Anti-neurofilament and anti-PGP9.5 visualized the overall innervation. Anti-MBP visualized myelination, anti-CGRP and anti-SP nociceptive fibers, anti-TH sympathetic nerve fibers, and anti-GAP43 nerve fibers during development and regeneration. Immunolabeled sections were analyzed in the confocal laser scanning microscope. We show that the LHBT contains unmyelinated as well as myelinated nerve fibers which group in nerve fascicles and follow blood vessels. Manny myelinated and unmyelinated axons exhibit molecular features of nociceptive nerve fibers. Another subpopulation of unmyelinated axons exhibits molecular characteristics of sympathetic nerve fibers. Unmyelinated sympathetic fibers and unmyelinated nociceptive fibers express proteins that are found during development and regeneration. Present findings support the hypothesis that ingrowth of nociceptive fibers are the source of chronic tendon pain.

Axonal components of nerves innervating the human arm.

OBJECTIVE: Axons traveling within the brachial plexus are responsible for the dexterous control of human arm and hand movements. Despite comprehensive knowledge on the topographical anatomy of nerves innervating the human upper limbs, the definite quantity of sensory and motor axons within this neural network remains elusive. Our aim was to perform a quantitative analysis of the axonal components of human upper limb nerves based on highly specific molecular features from spinal cord level to the terminal nerves at wrist level. METHODS: Nerve specimen harvest at predefined harvesting sites (plexus roots and cords as well as major nerves originating from the brachial plexus innervating the arm and hand) was performed in 9 human heart-beating organ donors. Double immunofluorescence staining using antibodies against choline-acetyltransferase and neurofilament was performed to differentiate motor and sensory axons on nerve cross sections. RESULTS: Three hundred fifty thousand axons emerge from the spinal cord to innervate the human upper limb, of which 10% are motor neurons. In all nerves studied, sensory axons outnumber motor axons by a ratio of at least 9:1. The sensory axon contribution increases when moving distally, whereas only 1,700 motor axons reach the hand to innervate the intrinsic musculature. INTERPRETATION: Our results suggest that upper limb motor execution, and particularly dexterous coordination of hand movement, require an unexpectedly low number of motor neurons, with a large convergence of afferent input for feedback control. Ann Neurol 2017;82:396-408.

The Regenerative Capability of the Urodele Amphibians and Its Potential for Plastic Surgery.

Newts and salamanders, both urodele amphibians, are the only vertebrates with tremendous regenerative potency throughout their lifetime. In contrast to the limited regenerative potential of most mammals, including humans, they can regenerate an entire limb after amputation and many other structures of their bodies, whereas humans mainly respond to injury by the formation of a scar. The intention of plastic surgery is to restore function of injured body parts, with the highest principle to replace "like with like." Despite tremendous improvements in surgical techniques over the last century, the remaining drawbacks include the availability of autologous tissue for transfer to restore extensive tissue loss. Here, some regenerative features of the urodeles are reviewed, in particular wound healing, nerve and limb regeneration, and their potential impact for reconstructive surgery are discussed. With a detailed molecular and cellular understanding of the urodele regeneration processes in combination with recent advances in tissue engineering, new perspectives for plastic surgery and especially improvements in regards to tissue regeneration are opened.

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Langerhans cell maturation is accompanied by induction of N-cadherin and the transcriptional regulators of epithelial-mesenchymal transition ZEB1/2.

Langerhans cells (LCs) are a unique subset of dendritic cells (DCs) that express epithelial adhesion molecules, allowing them to form contacts with epithelial cells and reside in epidermal/epithelial tissues. The dynamic regulation of epithelial adhesion plays a decisive role in the life cycle of LCs. It controls whether LCs remain immature and sessile within the epidermis or mature and egress to initiate immune responses. So far, the molecular machinery regulating epithelial adhesion molecules during LC maturation remains elusive. Here, we generated pure populations of immature human LCs in vitro to systematically probe for gene-expression changes during LC maturation. LCs down-regulate a set of epithelial genes including E-cadherin, while they upregulate the mesenchymal marker N-cadherin known to facilitate cell migration. In addition, N-cadherin is constitutively expressed by monocyte-derived DCs known to exhibit characteristics of both inflammatory-type and interstitial/dermal DCs. Moreover, the transcription factors ZEB1 and ZEB2 (ZEB is zinc-finger E-box-binding homeobox) are upregulated in migratory LCs. ZEB1 and ZEB2 have been shown to induce epithelial-to-mesenchymal transition (EMT) and invasive behavior in cancer cells undergoing metastasis. Our results provide the first hint that the molecular EMT machinery might facilitate LC mobilization. Moreover, our study suggests that N-cadherin plays a role during DC migration.

Nerve Transfers for Brachial Plexus Reconstruction in Patients over 60 Years.

Negative expectations regarding nerve reconstruction in the elderly prevail in the literature, but little is known about the effectiveness of nerve transfers in patients with brachial plexus injuries aged over 60 years. We present a series of five patients (1 female, 4 male) aged between 60 and 81 years (median 62.0 years) who underwent nerve reconstruction using multiple nerve transfers in brachial plexopathies. The etiology of brachial plexus injury was trauma (n = 2), or iatrogenic, secondary to spinal surgical laminectomy, tumor excision and radiation for breast cancer (n = 3). All but one patient underwent a one-stage reconstruction including neurolysis and extra-anatomical nerve transfer alone (n = 2) or combined with anatomical reconstruction by sural nerve grafts (n = 2). One patient underwent a two-stage reconstruction, which involved a first stage anatomical brachial plexus reconstruction followed by a second stage nerve transfer. Neurotizations were performed as double (n = 3), triple (n = 1) or quadruple (n = 1) nerve or fascicular transfers. Overall, at least one year postoperatively, successful results, characterized by a muscle strength of M3 or more, were restored in all cases, two patients even achieving M4 grading in the elbow flexion. This patient series challenges the widely held dogma that brachial plexus reconstruction in older patients will produce poor outcomes. Distal nerve transfers are advantageous as they shorten the reinnervation distance. Healthy, more elderly patients should be judiciously offered the whole spectrum of reconstructive methods and postoperative rehabilitation concepts to regain useful arm and hand function and thus preserve independence after a traumatic or nontraumatic brachial plexus injury.

miR-146a is differentially expressed by myeloid dendritic cell subsets and desensitizes cells to TLR2-dependent activation.

Langerhans cells (LCs) in epithelia and interstitial dendritic cells (intDCs) in adjacent connective tissues represent two closely related myeloid-derived DC subsets that exert specialized functions in the immune system and are of clinical relevance for cell therapy. Both subsets arise from monocyte-committed intermediates in response to tissue-associated microenvironmental signals; however, molecular mechanisms underlying myeloid DC subset specification and function remain poorly defined. Using microarray profiling, we identified microRNA (miRNA) miR-146a to be constitutively expressed at higher levels in human LCs compared with intDCs. Moreover, miR-146a levels were low in monocytes and nondetectable in neutrophil granulocytes. Interestingly, constitutive high miR-146a expression in LCs is induced by the transcription factor PU.1 in response to TGF-beta1, a key microenvironmental signal for epidermal LC differentiation. We identified miR-146a as a regulator of monocyte and DC activation but not myeloid/DC subset differentiation. Ectopic miR-146a in monocytes and intDCs interfered with TLR2 downstream signaling and cytokine production, without affecting phenotypic DC maturation. Inversely, silencing of miR-146a in LCs enhanced TLR2-dependent NF-kappaB signaling. We therefore conclude that high constitutive miR-146a levels are induced by microenvironmental signals in the epidermis and might render LCs less susceptible to inappropriate activation by commensal bacterial TLR2 triggers at body surfaces.

[Babysitter nerve transfer from the thenar branch to the deep terminal branch of the ulnar nerve : An option to preserve the intrinsic hand muscles in proximal lesions of the ulnar nerve]. / "Babysitter"-Nerventransfer vom R. thenaris zum R. profundus nervi ulnaris : Eine Option zum Erhalt der intrinsischen Handmuskulatur bei hohen Läsionen des N. ulnaris.

OBJECTIVE: The objective of this surgery is to achieve early reinnervation of the intrinsic hand muscles through axons of the median nerve, preventing irreversible atrophy of the muscle tissue. The nerve transfer is achieved via a babysitter graft, which is sutured end-to-side to the donor as well as the recipient nerve. The procedure is carried out in combination with a proximal reconstruction of the ulnar nerve. INDICATIONS: High-grade lesions of the ulnar nerve without spontaneous regeneration, particularly when lesions are located proximally and/or when patients present late. CONTRAINDICATIONS: Irreversible denervation of the intrinsic muscles; weakness or palsy of the thenar branch. SURGICAL TECHNIQUE: The approach is taken through a longitudinal incision over the volar wrist. The deep branch of the ulnar nerve as well as the thenar branch of the median nerve are visualized after transection of the flexor retinaculum. An autologous graft is then placed between the two nerves, sutured to the donor (thenar branch) as well as the recipient nerve (ulnar deep branch) via an epineural window in an end-to-side manner. This facilitates timely regeneration of motor axons from the median nerve into the intrinsic muscles, thereby preventing irreversible degeneration. Through the end-to-side nerve coaptation, damage to the donor nerve is reduced to a minimum. At the same time reconstruction of the ulnar nerve is performed proximally to the lesion, facilitating original reinnervation of the intrinsic muscles at a later time. POSTOPERATIVE MANAGEMENT: Postoperatively, Penrose drains are placed and a sterile hand dressing is applied. Drain removal and dressing change are performed on the first day, suture removal after 2 weeks. Physical therapy for mobility of the joints can be started as early as 1 week after surgery. After the first signs of motor and/or sensory reinnervation, a targeted retraining of daily skills should be initiated. RESULTS: This procedure has so far been reported on three patients with high-grade ulnar nerve injury. After a follow-up duration of 6 years, each achieved muscle strength of ≥ M3, with good to excellent overall regeneration according to the modified Bishop rating scale.

Molecular Pattern and Density of Axons in the Long Head of the Biceps Tendon and the Superior Labrum.

The type II superior labrum anterior to posterior (SLAP) repair is a viable option in young and demanding patients, although a prolonged period of pain after surgery is described in the literature. The reason for this fact remains unknown. Thus, the purpose of this study was to investigate the molecular pattern of the biceps tendon anchor, where the sutures for repair are placed. The long head of the biceps tendon (LHBT), including the superior labrum, was dissected in the setting of reverse total shoulder arthroplasty. Immunohistochemical staining was performed using neurofilament (NF) and protein gene product (PGP) 9.5 as general markers for axons and calcitonin gene-related peptide (CGRP) and substance P for nociceptive transmission. A quantitative assessment was performed according to the two regions of interest (ROIs), i.e., the anterosuperior (ROI I) and the posterosuperior labrum (ROI II). Eleven LHBTs with a mean age of 73 years (range: 66-87 years) were harvested intraoperatively. Six LHBTs were gained in osteoarthrosis and five in fractures. We found an inhomogeneous distribution of axons in the anterosuperior and posterosuperior parts of the labrum in all the specimens irrespective of the age, gender, and baseline situation. There was a significantly higher number (p < 0.01) as well as density (p < 0.001) of NF-positive axons in ROI I compared to ROI II. Nociceptive fibers were always found along the NF-positive axons. Thus, our results indicate that the biceps tendon anchor itself is a highly innervated region comprising different nerve qualities. The anterosuperior labrum contains a higher absolute number and density of axons compared to the posterosuperior parts. Furthermore, we were able to prove the presence of nociceptive fibers in the superior labrum. The results obtained in this study could contribute to the variability of pain after SLAP repair.

How to visualize the innervation pattern in tendons: A methodical guide.

BACKGROUND: Tendon pathologies are common and several data suggests that the peripheral nervous system is involved in this disorder. Immunohistochemistry (IHC) is one of the pillars to characterize nervous structures and their implication in the pathogenesis of chronic tendon pain. Most commonly, formalin-fixed, paraffin-embedded (FFPE) tendons are used for immunohistochemical characterization of the innervation. However, FFPE specimens exhibit major disadvantages: First, antigens (proteins) are masked and antigen retrieval is necessary to restore antigenicity. Second, FFPE specimens involve immunolabeling with enzyme-conjugated antibodies but this approach has limitations when multiple antigens are of interest simultaneously. Consequently, there is a demand in the orthopedic community for an alternative immunohistochemical approach to visualize tendon innervations. RESULTS: Here, we present a guide how to visualize tendon innervation. This guide couples paraformaldehyde fixation, cryo-embedding, immunofluorescence, and confocal laser scanning microscopy. We demonstrate the utility of our approach in the long head of the biceps tendon. For nerve fiber characterization, we used different neuronal markers including antibodies against neurofilament, protein gene product 9.5, calcitonin gene related peptide, and substance P. We show that it is possible to collect high quality, multicolor images of the innervation pattern of tendons. To map immunolabeled structures and the anatomical structures of the tendon fluorescence images and bright field images were merged. CONCLUSION: For the orthopedic community our approach might be a convenient research tool to simultaneously utilize multiple neuronal markers on the same tissue section and to define with greater accuracy the heterogeneity of tendon innervation.

Lineage Identity and Location within the Dermis Determine the Function of Papillary and Reticular Fibroblasts in Human Skin.

Human skin dermis is composed of the superficial papillary dermis and the reticular dermis in the lower layers, which can easily be distinguished histologically. In vitro analyses of fibroblasts from explant cultures from superficial and lower dermal layers suggest that human skin comprises at least two fibroblast lineages with distinct morphology, expression profiles, and functions. However, while for mouse skin cell surface markers have been identified, allowing the isolation of pure populations of one lineage or the other via FACS, this has not been achieved for human skin fibroblasts. We have now discovered two cell surface markers that discriminate between papillary and reticular fibroblasts. While FAP+CD90- cells display increased proliferative potential, express PDPN and NTN1, and cannot be differentiated into adipocytes, FAP-CD90+ fibroblasts express high levels of ACTA2, MGP, PPARγ, and CD36 and readily undergo adipogenic differentiation, a hallmark of reticular fibroblasts. Flow cytometric analysis of fibroblasts isolated from superficial and lower layers of human dermis showed that FAP+CD90- cells are enriched in the papillary dermis. Altogether, functional analysis and expression profiling confirms that FAP+CD90- cells represent papillary fibroblasts, whereas FAP-CD90+ fibroblasts derive from the reticular lineage. Although papillary and reticular fibroblasts are enriched in the upper or lower dermis, respectively, they are not spatially restricted, and the microenvironment seems to affect their function.

Palisade Endings Are a Constant Feature in the Extraocular Muscles of Frontal-Eyed, But Not Lateral-Eyed, Animals.

PURPOSE: To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). METHODS: Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. RESULTS: In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin. CONCLUSIONS: Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence.

Identification of Axl as a downstream effector of TGF-ß1 during Langerhans cell differentiation and epidermal homeostasis.

Transforming growth factor-ß1 (TGF-ß1) is a fundamental regulator of immune cell development and function. In this study, we investigated the effects of TGF-ß1 on the differentiation of human Langerhans cells (LCs) and identified Axl as a key TGF-ß1 effector. Axl belongs to the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, whose members function as inhibitors of innate inflammatory responses in dendritic cells and are essential to the prevention of lupus-like autoimmunity. We found that Axl expression is induced by TGF-ß1 during LC differentiation and that LC precursors acquire Axl early during differentiation. We also describe prominent steady-state expression as well as inflammation-induced activation of Axl in human epidermal keratinocytes and LCs. TGF-ß1-induced Axl enhances apoptotic cell (AC) uptake and blocks proinflammatory cytokine production. The antiinflammatory role of Axl in the skin is reflected in a marked impairment of the LC network preceding spontaneous skin inflammation in mutant mice that lack all three TAM receptors. Our findings highlight the importance of constitutive Axl expression to tolerogenic barrier immunity in the epidermis and define a mechanism by which TGF-ß1 enables silent homeostatic clearing of ACs to maintain long-term self-tolerance.

Identification of TROP2 (TACSTD2), an EpCAM-like molecule, as a specific marker for TGF-ß1-dependent human epidermal Langerhans cells.

Langerin (CD207) expression is a hallmark of epidermal Langerhans cells (LCs); however, CD207(+) cells comprise several functional subsets. Murine studies showed that epidermal, but not dermal, CD207(+) cells require transforming growth factor-ß 1 (TGF-ß1) for development, whereas human data are lacking. Using gene profiling, we found that the surface molecule TROP2 (TACSTD2) is strongly and rapidly induced during TGF-ß1-dependent LC commitment of human CD34(+) hematopoietic progenitor cells or monocytes. TROP2 is conserved between mouse and human, and shares substantial amino-acid identity with EpCAM, a marker for murine epidermal LCs. To our knowledge, neither TROP2 nor EpCAM expression has been analyzed in human dendritic cell (DC) subsets. We found that (i) all human epidermal LCs are TROP2(+)EpCAM(+); (ii) human dermis lacks CD207(+)EpCAM(-) or CD207(+)TROP2(-) DCs, i.e., equivalents of murine dermal CD207(+) DCs; and (iii) pulmonary CD207(+) cells are TROP2(-)EpCAM(-). Moreover, although EpCAM was broadly expressed by pulmonary and intestinal epithelial cells, as well as by bone marrow erythroid progenitor cells, these cells lacked TROP2. However, although TROP2 is expressed by human LCs as well as by human and murine keratinocytes, most murine LCs, except of a small subset, lacked TROP2. Therefore, TROP2 is a marker for human TGF-ß1-dependent epidermal LCs.