Preoperative estimation of tibial nail length--because size does matter.

INTRODUCTION: Selecting the correct tibial nail length is essential for satisfactory outcomes. Nails that are inserted and are found to be of inappropriate length should be removed. Accurate preoperative nail estimation has the potential to reduce intra-operative errors, operative time and radiation exposure. METHODS: We compared the most commonly used radiological, anthropometric and intra-operative techniques to determine ideal nail lengths for 16 paired cadaveric tibiae. Five different anthropometric measurements were taken from each intact cadaver including: knee joint line to ankle joint line distance (JJD), medial knee joint line to medial malleolus distance (MMD), tibial tuberosity to medial malleolus distance (TMD), olecranon to 5th metacarpal head distance (OMD) and body height (BHR). Each tibia also underwent antero-posterior (AP) and lateral scanograms. Computerised tomography was used to determine the ideal nail length for each tibia. Each anthropometric and radiological measurement was recorded by two orthopaedic surgeons independently. An expert tibial nail was then inserted after nail length estimation was performed using a guidewire technique and an intra-operative radiographic ruler. RESULTS: The AP scanogram was found to be 100% accurate in selecting ideal nail length. The lateral scanogram was also found to be reasonably accurate but in 19% (3/16) of cases it led to a nail being too long. The intra-operative radiographic ruler was found to give a good indication of the ideal nail size, as did the guidewire technique, with only 6% (1/16) of cases producing an incorrect nail size. In general, the anatomical measurements gave a poor indication of ideal nail size compared with the other techniques. The following accuracies were noted: JJD 56%, MMD 50%, TMD 38%, BHR 13% and OMD 56%. CONCLUSIONS: We found that radiological methods such as using an AP radiograph with known magnification and intra-operative radiographic ruler were able to predict nail length very accurately and we suggest that these measurements should be performed routinely. The guidewire technique was also effective but we recommend that it not be used in isolation as errors can occur. We found that anatomical measurements are not accurate for predicting tibial nail length.

Initial experience of double-layer tension free reconstruction of abdominal wall defects with porcine acellular dermal collagen implant and polypropylene mesh.

INTRODUCTION AND AIM: Various techniques have been proposed for the repair of abdominal wall defects (AWD) with varying rates of success. Despite the development of new materials and modifications of surgical technique, no single approach has emerged as the optimum way to close large AWD. We report a method for repairing large incisional and recurrent abdominal wall hernias using a double-mesh technique. The defect is closed using an underlay biological implant and an onlay synthetic mesh, which is 'quilted' to the underlying abdominal wall and biological implant. The current study reports our initial experience with this approach in ten consecutive patients operated on for large AWD. METHODS: In this prospective observational study the following data were collected: age, gender, previous surgery, co-morbidities, situation and size of the defect, antibiotic therapy, hospital stay, postoperative complications and bacteriology in case of infection. The patients were reviewed at 1, 3 and 6 months, and 1-year postsurgery. RESULTS: Overall all ten AWD of ≥ 75 cm(2) were reconstructed successfully using the quilting technique. Median age of patients was 61 years (range 47-73 years); male:female ratio was 3:2 and median weight was 107.5 kg. Two patients developed a wound infection and were treated successfully with antibiotics. At median follow-up of 15.5 months (range 6-29 months) there was no case of recurrence. CONCLUSION: The use of double-layer of porcine acellular dermal collagen implant and polypropylene mesh in reconstruction of AWD can be considered a safe and effective treatment. The early short-term results are encouraging with few complications.

Critical roles for the netrin receptor deleted in colorectal cancer in dopaminergic neuronal precursor migration, axon guidance, and axon arborization.

DCC (deleted in colorectal cancer), a receptor for the axon guidance cue netrin-1, is highly expressed by mesencephalic dopaminergic (DA) neurons during development; however, the contribution of DCC to DA development remains largely uncharacterized. DA neurons in ventral mesencephalic nuclei also express UNC5 homologue netrin receptors from late embryogenesis to adulthood, raising the possibility that DA axons could be attracted or repelled by netrins. Examining newborn dcc null mice, we report that loss of DCC function results in profound alterations of DA circuitry, including DA progenitor cell migration defects, reduced numbers of DA cells in midbrain nuclei, an anomalous DA ventral commissure, malformed DA innervation of the ventral striatum, and reduced DA innervation of the cerebral cortex. Caspase-3 activation was detected in inappropriately localized DA cells, consistent with apoptosis contributing to reduced cell numbers. Dcc heterozygous mice express reduced levels of DCC protein. Although less severely disrupted than dcc nulls, newborn and adult dcc heterozygotes also have fewer DA neurons in ventral mesenscephalic nuclei. Despite the reduced numbers of DA neurons, newborn dcc heterozygotes and nulls exhibit similar DA innervation density as wild-type littermates in the nucleus accumbens core, and adult dcc heterozygotes exhibit increased DA innervation in medial prefrontal cortex. A trend towards increased innervation of medial prefrontal cortex was detected in newborn dcc heterozygotes, but did not reach statistical significance, suggesting that the increase in adult heterozygotes results from enhanced DA arborization during postnatal development. Consistent with the hypothesis that DCC regulates DA axonal projections, disrupting DCC function in culture inhibits netrin-1 induced DA axon extension and axon branching. Furthermore, disrupting DCC function in isolated DA neurons grown as micro-island cultures reduces the number of autaptic synapses per cell. We conclude that DCC regulates appropriate precursor cell migration, axon guidance, and terminal arborization by DA neurons.

Graded expression of netrin-1 by specific neuronal subtypes in the adult mammalian striatum.

Netrins are a family of secreted proteins that function as axon guidance cues during neural development. High levels of netrin-1 expressed by the embryonic ganglionic eminence, the precursor of the adult striatum, direct axons that pioneer the internal capsule. Here we describe netrin-1 expression by neurons distributed throughout the striatum of the adult mouse. Differences were detected in the number and type of neurons expressing netrin-1 in different regions of the striatum. Most neurons in the ventral striatum, including projection neurons and cholinergic interneurons, express netrin-1. In contrast, netrin-1 expression is largely limited to cholinergic interneurons in the dorsal striatum, and the proportion of cholinergic interneurons that express netrin-1 decreases along rostral-caudal and ventral-dorsal axes. Regional differences in expression in the adult striatum suggest that netrin-1 not only influences the development of striatal circuitry but may also participate in the maintenance and plasticity of connections in the adult brain.

Netrin receptor deficient mice exhibit functional reorganization of dopaminergic systems and do not sensitize to amphetamine.

Netrins are guidance cues that play a fundamental role in organizing the developing brain. The netrin receptor, DCC (deleted in colorectal cancer), is highly expressed by dopaminergic (DA) neurons. DCC may therefore participate in the organization of DA circuitry during development and also influence DA function in the adult. Here we show that adult dcc heterozygous mice exhibit a blunted behavioral response to the indirect DA agonist amphetamine and do not develop sensitization to its effects when treated repeatedly. These behavioral alterations are associated with profound changes in DA function. In the medial prefrontal cortex, dcc heterozygotes exhibit increased tyrosine hydroxylase (TH) protein levels and dramatic increases in basal concentrations of DA and DA metabolites. In contrast, in the nucleus accumbens, dcc heterozygotes show no changes in either TH or DA levels, but exhibit decreased concentrations of DA metabolites, suggesting reduced DA activity. In addition, dcc heterozygous mice exhibit a small, but significant reduction in total number of TH-positive neurons in midbrain DA cell body regions. These results demonstrate for the first time that alterations in dcc expression lead to selective changes in DA function and, in turn, to differences in DA-related behaviors in adulthood. These findings raise the possibility that changes in dcc function early in life are implicated in the development of DA dysregulation observed in certain psychiatric disorders, such as schizophrenia, or following chronic use of drugs of abuse.

Widespread expression of netrin-1 by neurons and oligodendrocytes in the adult mammalian spinal cord.

Netrins are a family of secreted proteins that function as chemotropic axon guidance cues during neural development. Here we demonstrate that netrin-1 continues to be expressed in the adult rat spinal cord at a level similar to that in the embryonic CNS. In contrast, netrin-3, which is also expressed in the embryonic spinal cord, was not detected in the adult. In situ hybridization analysis demonstrated that cells in the white matter and the gray matter of the adult spinal cord express netrin-1. Colocalization studies using the neuronal marker NeuN revealed that netrin-1 is expressed by multiple classes of spinal interneurons and motoneurons. Markers identifying glial cell types indicated that netrin-1 is expressed by most, if not all, oligodendrocytes but not by astrocytes. During neural development, netrin-1 has been proposed to function as a diffusible long-range cue for growing axons. We show that in the adult spinal cord the majority of netrin-1 protein is not freely soluble but is associated with membranes or the extracellular matrix. Fractionation of adult spinal cord white matter demonstrated that netrin-1 was absent from fractions enriched for compact myelin but was enriched in fractions containing periaxonal myelin and axolemma, indicating that netrin-1 protein may be localized to the periaxonal space. These findings suggest that in addition to its role as a long-range guidance cue for developing axons, netrin may have a short-range function associated with the cell surface that contributes to the maintenance of appropriate neuronal and axon-oligodendroglial interactions in the mature nervous system.

The p75 neurotrophin receptor activates Akt (protein kinase B) through a phosphatidylinositol 3-kinase-dependent pathway.

The Akt kinase plays a crucial role in supporting Trk-dependent cell survival, whereas the p75 neurotrophin receptor (p75NTR) facilitates cellular apoptosis. The precise mechanism that p75NTR uses to promote cell death is not certain, but one possibility is that p75NTR-dependent ceramide accumulation inhibits phosphatidylinositol 3-kinase-mediated Akt activation. To test this hypothesis, we developed a system for examining p75NTR-dependent apoptosis and determined the effect of p75NTR on Akt activation. Surprisingly, p75NTR increased, rather than decreased, Akt phosphorylation in a variety of cell types, including human Niemann-Pick fibroblasts, which lack acidic sphingomyelinase activity. The p75NTR expression level required to elicit Akt phosphorylation was much lower than that required to activate the JNK pathway or to mediate apoptosis. We show that p75NTR-dependent Akt phosphorylation was independent of TrkA signaling, required active phosphatidylinositol 3-kinase, and was associated with increased tyrosine phosphorylation of p85 and Shc and with reduced cytosolic tyrosine phosphatase activity. Finally, we show that p75NTR expression increased survival in cells exposed to staurosporine or subjected to serum withdrawal. These findings indicate that p75NTR facilitates cell survival through novel signaling cascades that result in Akt activation.

Expression of netrin-1 and its receptors DCC and UNC-5H2 after axotomy and during regeneration of adult rat retinal ganglion cells.

Netrins are a family of chemotropic factors that guide axon outgrowth during development; however, their function in the adult CNS remains to be established. We examined the expression of the netrin receptors DCC and UNC5H2 in adult rat retinal ganglion cells (RGCs) after grafting a peripheral nerve (PN) to the transected optic nerve and following optic nerve transection alone. In situ hybridization revealed that both Dcc and Unc5h2 mRNAs are expressed by normal adult RGCs. In addition, netrin-1 was found to be constitutively expressed by RGCs. Quantitative analysis using in situ hybridization demonstrated that both Dcc and Unc5h2 were down-regulated by RGCs following axotomy. In the presence of an attached PN graft, Dcc and Unc5h2 were similarly down-regulated in surviving RGCs regardless of their success in regenerating an axon. Northern blot analysis demonstrated expression of netrin-1 in both optic and sciatic nerve, and Western blot analysis revealed the presence of netrin protein in both nerves. Immunohistochemical analysis indicated that netrin protein was closely associated with glial cells in the optic nerve. These results suggest that netrin-1, DCC, and UNC5H2 may contribute to regulating the regenerative capacity of adult RGCs.

Cellular mechanisms of netrin function: long-range and short-range actions.

Netrins are secreted proteins that direct axon extension and cell migration during neural development. They are bifunctional cues that act as an attractant for some cell types and as a repellent for others. Several lines of evidence suggest that two classes of receptors, the deleted in colorectal cancer (DCC) family and the UNC-5 family, mediate the attractant and repellent response to netrin. Although netrins were first identified as diffusible long-range cues for developing axons, recent findings provide evidence that they also function as short-range cues close to the surface of the cells that produce them. This short-range function of netrin contributes to guiding neurite outgrowth and mediating cell-cell interactions during development and perhaps also in adults.

Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.

Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase sigma.

Protein tyrosine phosphatase sigma (PTP-sigma, encoded by the Ptprs gene) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases that is highly expressed during mammalian embryonic development in the germinal cell layer lining the lateral ventricles of the developing brain, dorsal root ganglia, Rathke's pouch, olfactory epithelium, retina and developing lung and heart. On the basis of its expression and homology with the Drosophila melanogasterorthologues DPTP99 and DPTP100A (refs 5,6), which have roles in the targeting of axonal growth cones, we hypothesized that PTP-sigma may also have a modulating function in cell-cell interactions, as well as in axon guidance during mammalian embryogenesis. To investigate its function in vivo, we generated Ptprs-deficient mice. The resulting Ptprs-/-animals display retarded growth, increased neonatal mortality, hyposmia and hypofecundity. Anatomical and histological analyses showed a decrease in overall brain size with a severe depletion of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cells in Ptprs-/- hypothalamus. Ptprs-/- mice have an enlarged intermediate pituitary lobe, but smaller anterior and posterior lobes. These results suggest that tyrosine phosphorylation-dependent signalling pathways regulated by PTP-sigma influence the proliferation and/or adhesiveness of various cell types in the developing hypothalamo-pituitary axis.

A role for netrin-1 in the guidance of cortical efferents.

An intermediate target for axons leaving the cerebral cortex in embryonic mammals is the ganglionic eminence (GE), the embryonic precursor of the basal ganglia. The cues that direct these axons over the initial portion of their trajectory are not well understood, but could include both short-range and long-range attractants and repellents. In the present study, we provide evidence that corticofugal axons might be guided at least partly by a diffusible factor or factors originating in the lateral GE and the sulcus between the lateral and medial ridges of the GE (ISS), as well as evidence implicating the axonal chemoattractant netrin-1 in mediating these effects. Explants of lateral GE and ISS obtained from E12.5 and E13.5 mouse forebrain have a strong effect on both the outgrowth and orientation of corticofugal axons when cultured at a distance with explants of embryonic cortex in collagen gels. Netrin-1 mRNA is detected in these target tissues by in situ hybridization, and both netrin-1 protein and heterologous cells secreting netrin-1 can mimic the outgrowth-promoting effect of these target tissues in vitro. Furthermore, the growth of corticofugal axons is oriented toward an ectopic source of netrin-1 in vitro, and a function blocking anti-netrin-1 antiserum specifically abolishes the cortical axon outgrowth elicited by explants of lateral GE and the ISS in collagen gel cocultures. Taken together, these results suggest a role for netrin-1 in the attraction at a distance of early cortical axons by the GE. Thus in mammals -- as is also observed in nematodes -- the development of non-commissural projections in anterior regions of the embryo might be directed by mechanisms similar to those involved in directing the development of commissural projections in more posterior regions of the central nervous system.

Netrin-1 and DCC mediate axon guidance locally at the optic disc: loss of function leads to optic nerve hypoplasia.

Embryonic retinal ganglion cell (RGC) axons must extend toward and grow through the optic disc to exit the eye into the optic nerve. In the embryonic mouse eye, we found that immunoreactivity for the axon guidance molecule netrin-1 was specifically on neuroepithelial cells at the disk surrounding exiting RGC axons, and RGC axons express the netrin receptor, DCC (deleted in colorectal cancer). In vitro, anti-DCC antibodies reduced RGC neurite outgrowth responses to netrin-1. In netrin-1- and DCC-deficient embryos, RGC axon pathfinding to the disc was unaffected; however, axons failed to exit into the optic nerve, resulting in optic nerve hypoplasia. Thus, netrin-1 through DCC appears to guide RGC axons locally at the optic disc rather than at long range, apparently reflecting the localization of netrin-1 protein to the vicinity of netrin-1-producing cells at the optic disc.

Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons.

Two tandem Netrin genes in Drosophila are expressed at the midline of the developing CNS and in different subsets of neurons, muscles, and epidermal patches. In embryos carrying a small deficiency that deletes both genes, CNS axon commissures are partially missing or thinner. This phenotype is rescued by expressing either Netrin gene at the midline. Pan-neural expression of either gene causes disruption of commissural and longitudinal tracts, indicating that the pattern of Netrin expression is crucial and that Netrins function as instructive cues. The double mutant also shows defects in motor axon projections. Expression of either Netrin gene in all muscles also results in aberrant motor projections. Thus, Drosophila Netrins are required for the guidance of commissural axons at the midline, and of motor axons to their target muscles.

Guidance and induction of branch formation in developing axons by target-derived diffusible factors.

Developing axons appear to navigate to their targets partly in response to long-range diffusible chemoattractants secreted by target cells. The past year has seen the identification of the first target-derived attractants for developing axons, the netrins. Target-derived diffusible factors have also been implicated in controlling the elaboration of axon collateral branches, and a recent study provides insight into the mechanism by which this is achieved. Evidence has also suggested neurotrophins and neurotransmitters as candidate branching and chemotropic factors.

Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.

The guidance of axons to their targets in the developing nervous system is believed to involve diffusible chemotropic factors secreted by target cells. Floor plate cells at the ventral midline of the spinal cord secrete a diffusible factor or factors that promotes the outgrowth of spinal commissural axons and attracts these axons in vitro. Two membrane-associated proteins isolated from brain, netrin-1 and netrin-2, possess commissural axon outgrowth-promoting activity. We show here that netrin-1 RNA is expressed by floor plate cells, whereas netrin-2 RNA is detected at lower levels in the ventral two-thirds of the spinal cord, but not the floor plate. Heterologous cells expressing recombinant netrin-1 or netrin-2 secrete diffusible forms of the proteins and can attract commissural axons at a distance. These results show that netrin-1 is a chemotropic factor expressed by floor plate cells and suggest that the two netrin proteins guide commissural axons in the developing spinal cord.

The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.

In vertebrates, commissural axons pioneer a circumferential pathway to the floor plate at the ventral midline of the embryonic spinal cord. Floor plate cells secrete a diffusible factor that promotes the outgrowth of commissural axons in vitro. We have purified from embryonic chick brain two proteins, netrin-1 and netrin-2, that each possess commissural axon outgrowth-promoting activity, and we have also identified a distinct activity that potentiates their effects. Cloning of cDNAs encoding the two netrins shows that they are homologous to UNC-6, a laminin-related protein required for the circumferential migration of cells and axons in C. elegans. This homology suggests that growth cones in the vertebrate spinal cord and the nematode are responsive to similar molecular cues.