Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitallv at 2 mm. (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast-mineralized matrix complex. Histologically, at this time point a tissue structure with a connective tissue-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of inummodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Feedforward activity of the cervical flexor muscles during voluntary arm movements is delayed in chronic neck pain

The objective of this study was to compare onset of deep and superficial cervical flexor muscle activity during rapid, unilateral arm movements between ten patients with chronic neck pain and 12 control subjects. Deep cervical flexor (DCF) electromyographic activity (EMG) was recorded with custom electrodes inserted via the nose and fixed by suction to the posterior mucosa of the oropharynx. Surface electrodes were placed over the sternocleidomastoid (SCM) and anterior scalene (AS) muscles. While standing, subjects flexed and extended the right arm in response to a visual stimulus. For the control group, activation of DCF, SCM and AS muscles occurred less than 50 ms after the onset of deltoid activity, which is consistent with feedforward control of the neck during arm flexion and extension. When subjects with a history of neck pain flexed the arm, the onsets of DCF and contralateral SCM and AS muscles were significantly delayed (p<0.05). It is concluded that the delay in neck muscle activity associated with movement of the arm in patients with neck pain indicates a significant deficit in the automatic feedforward control of the cervical spine. As the deep cervical muscles are fundamentally important for support of the cervical lordosis and the cervical joints, change in the feedforward response may leave the cervical spine vulnerable to reactive forces from arm movement.

Etr1-1 gene expression alters regeneration patterns in transgenic lettuce stimulating root formation

We have evaluated the transformation efficiency of two lettuce ( Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens- mediated gene transfer. Six- day- old cotyledons were co- cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the beta- glucuronidase gene ( GUS) under the control of the cauliflower mosaic virus 35S promoter ( CaMV 35S), while the second construct contained the ethylene mutant receptor etr1- 1, which confers ethylene insensitivity, under the control of a leaf senescence- specific promoter ( sag12). Tissues co- cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were transgenic. Very different results were observed when cotyledon explants were co- cultivated with Agrobacteria carrying the etr1- 1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed in 10% of cotyledons and some organogenic shoots were obtained ( 2.86%). These results indicate that the ethylene insensitivity conferred by the etr1- 1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating root formation during regeneration.

The traditional rationale of the arm's length approach to transfer pricing - should the separate accounting model be maintained for modern multinational entities?

This article examines the current transfer pricing regime to consider whether it is a sound model to be applied to modern multinational entities. The arm's length price methodology is examined to enable a discussion of the arguments in favour of such a regime. The article then refutes these arguments concluding that, contrary to the very reason multinational entities exist, applying arm's length rules involves a legal fiction of imagining transactions between unrelated parties. Multinational entities exist to operate in a way that independent entities would not, which the arm's length rules fail to take into account. As such, there is clearly an air of artificiality in applying the arm's length standard. To demonstrate this artificiality with respect to modern multinational entities, multinational banks are used as an example. The article concluded that the separate entity paradigm adopted by the traditional transfer pricing regime is incongruous with the economic theory of modern multinational enterprises.

Functionalized macrocycles from functionalized tetra-amines: Pendent-arm macrocycles derived from dichloropivalic acid

Reaction between ethane-1,2-diamine and 3,3'-dichloropivalic acid results in different, isomeric tetra-amine derivatives, one a tetraamino carboxylic acid and the other a carboxamidotriamino alcohol, depending upon reaction conditions, Intended conversion of the Cu(II) complex of the former to a cyclam-like macrocycle through reaction with nitroethane and formaldehyde results in isolation of derivatives of both the former and the latter. This can be rationalized by assuming the intermediacy of an azetidinone, a species similar to that seen in simpler reactions of dichloropivalates. A single reaction thereby provides pendent-arm macrocycles where one has an electrophilic and the other a nucleophilic substituent. Parallel chemistry is not seen in the reaction between propane-1,3-diamine and 3,3'-dichloropivalate.

Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration

The muscle isoform. of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice

Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that adult mice lacking EphA4 (-/-), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal regeneration and functional recovery after spinal cord hemisection. Anterograde and retrograde tracing showed that axons from multiple pathways, including corticospinal and rubrospinal tracts, crossed the lesion site. EphA4 -/- mice recovered stride length, the ability to walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1-3 months of injury. EphA4 expression was upregulated on astrocytes at the lesion site in wild-type mice, whereas astrocytic gliosis and the glial scar were greatly reduced in lesioned EphA4-/- spinal cords. EphA4 -/- astrocytes failed to respond to the inflammatory cytokines, interferon-gamma or leukemia inhibitory factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4 -/- astrocytes, but longer neurites when the astrocyte EphA4 was blocked by monomeric EphrinA5-Fc. Thus, EphA4 regulates two important features of spinal cord injury, axonal inhibition, and astrocytic gliosis.

Pathways to improving skin regeneration

A significant proportion of the human population suffers from some form of skin disorder, whether it be from burn injury or inherited skin anomalies. The ideal treatment for skin disorders would be to regrow skin tissue from stem cells residing in the individual patient's skin. Locating these adult stem cells and elucidating the molecules involved in orchestrating the production of new skin cells are important steps in devising more-efficient methods of skin production and wound healing via the ex vivo expansion of patient keratinocytes in culture. This review focuses on the structure of the skin, the identification of skin stem cells, and the role of Notch, Wnt and Hedgehog signalling cascades in regulating the fate of epidermal stem cells. © 2005 Cambridge University Press.