Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats.

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex(®), a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex(®) fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex(®) fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex(®) fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex(®) conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex(®) conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex(®) conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.

Differential effects of riluzole on subpopulations of adult rat dorsal root ganglion neurons in vitro.

Riluzole is clinically approved for the treatment of motoneuron disease. We have previously shown that this drug is neuroprotective for both sensory neurons and motoneurons and promotes neurite outgrowth [Bergerot A, Shortland PJ, Anand P, Hunt SP, Carlstedt T (2004) Exp Neurol 187(2):359-366; Shortland PJ, Leinster VH, White W, Robson LG (2006) Eur J Neurosci 24:3343-3353]. This study explored the effects of exogenous administration of 0.1 muM riluzole on the neurite growth of specific subpopulations of adult rat dorsal root ganglion (DRG) neurons in vitro. Neuronal branching and neurite length were measured in calcitonin gene related peptide (CGRP), Griffonia simplicifolia Isolectin B4 (IB4), N52 and parvalbumin positive neuronal subpopulations. Riluzole was found to enhance neurite branching in both CGRP and IB4 positive neurons compared to vehicle treated cultures. However, neurite length was only significantly increased in CGRP positive neurons in riluzole treated cultures. The results suggest that riluzole affects specific subpopulations of sensory neurons in vitro and that its effects may be mediated through activation of neurotrophic factor receptors, since neurite outgrowth could be blocked by the administration of K252a (at 10 nM). Riluzole may offer a new pharmacological approach to promote sensory regeneration following small fibre neuropathies.

Neuronal calcium sensor-1 is expressed by dorsal root ganglion cells, is axonally transported to central and peripheral terminals, and is concentrated at nodes.

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily which has been implicated in the modulation of a number of neuronal functions. In this study we have examined the expression of NCS-1 in adult rat dorsal root ganglion (DRG) neurons. NCS-1 immunoreactivity was present in most DRG neurons, including many calcitonin gene-related peptide (CGRP) expressing ones. NCS-1 showed some colocalization with the synaptic vesicle protein synaptophysin and underwent both anterograde and retrograde axonal transport. NCS-1 immunoreactivity was also present in the dorsal horn of the spinal cord, and in peripheral cutaneous terminals innervating blood vessels, where it was coexpressed with CGRP. In addition, NCS-1 in peripheral nerves was concentrated at nodes and adjoining paranodes. These results suggest novel roles for NCS-1, particularly in relation to channel function at nodes and to the peripheral release of vasoactive peptides.

Comparison of metaphase and interphase FISH monitoring of minimal residual disease with MLL gene probe: case study of AML with t(9;11).

The place of FISH in the monitoring of minimal residual disease (MRD) is yet to be fully characterised. Routine bone marrow cytogenetics at diagnosis in a 22 year old patient with acute myeloid leukemia FAB type M5 detected a translocation t(9;11)(p22;q23). We report our investigations to assess residual levels of translocation using a FISH probe designed to detect a gene split by the translocation. We used MLL (Oncor), a probe which spans the MLL gene at 11q23, in both metaphase and interphase preparations. At diagnosis, metaphase FISH showed 3 distinct cell lines-normal with 2 signals, abnormal with 3 signals and abnormal with 2 signals, while interphase FISH showed only 2 cell lines, one with 2 signals (which could be normal or abnormal) and one with 3 signals (split MLL). Following treatment, with the patient in clinical remission, 7 further cytogenetic analyses and 2 further FISH analyses were compared. Our results suggest that monitoring of the t(9;11) by metaphase FISH is feasible and straightforward compared to cytogenetics but interphase FISH may be problematic.

Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts.

Patterning of fast and slow muscle fibres in limbs is regulated by signals from non-muscle cells. Myoblast lineage has, however, also been implicated in fibre type patterning. Here we test a founder cell hypothesis for the role of myoblast lineage, by implanting characterized fast and slow mouse myoblast clones into chick limb buds. In culture, late foetal mouse myoblast clones are committed to a probability (range 0-0.92) of slow myosin heavy chain (MyHC) expression. In contrast, when implanted into chick limbs, fast mouse myoblast clones express myosin characteristic of their new environment, without fusion to chick muscle cells and in the absence of innervation. Therefore, local signals exist within the chick limb bud during primary myogenesis that can override intrinsic commitment of at least some myoblasts, and induce slow MyHC.

Application of interphase FISH on direct bone marrow smears for evidence of chimerism in pediatric sex mismatched bone marrow transplantation.

We report here our experience in the management of four children undergoing sex mismatched allogeneic bone marrow transplantation (BMT) who showed evidence of poor engraftment post BMT. Fluorescence in situ hybridisation (FISH) was performed on direct bone marrow smears with the X centromere and Y heterochromatin probes, to ascertain chimerism. Of 16 hybridisations, only one was unsuccessful, whereas routine bone marrow cytogenetics performed at the same time failed in five of eight cultures. The FISH results were available in one to two days. The FISH findings provided a valuable aid in the early management of these children.

The distal limb environment regulates MyoD accumulation and muscle differentiation in mouse-chick chimaeric limbs.

Differentiation of muscle and cartilage within developing vertebrate limbs occurs in a proximodistal progression. To investigate the cues responsible for regulating muscle pattern, mouse myoblasts were implanted into early chick wings prior to endogenous chick muscle differentiation. Fetal myogenic cells originating from transgenic mice carrying a lacZ reporter were readily detected in vivo after implantation and their state of differentiation determined with species-specific antibodies to MyoD and myosin heavy chain. When mouse myogenic cells are implanted at the growing tip of early stage 21 limbs MyoD expression is suppressed and little differentiation of the mouse cells is detected initially. At later stages ectopically implanted mouse cells come to lie within muscle masses, re-express MyoD and differentiate in parallel with differentiating chick myoblasts. However, if mouse cells are implanted either proximally at stage 21 or into the limb tip at stage 24, situations in which mouse cells encounter endogenous differentiating chick myoblasts earlier, MyoD suppression is not detected and a higher proportion of mouse cells differentiate. Mouse cells that remain distal to endogenous differentiating myogenic cells are more likely to remain undifferentiated than myoblasts that lie within differentiated chick muscle. Undifferentiated distal mouse cells are still capable of differentiating if explanted in vitro, suggesting that myoblast differentiation is inhibited in vivo. In vitro, MyoD is suppressed in primary mouse myoblasts by the addition of FGF2 and FGF4 to the culture media. Taken together, our data suggest that the inhibition of myogenic differentiation in the distal limb involves MyoD suppression in myoblasts, possibly through an FGF-like activity.

Tissue and cellular patterning of the musculature in chick wings.

Development of the musculature involves generation of a precise number of individual muscles arranged in appropriate locations, each with the correct cellular patterning. To find out the rules that govern muscle number and arrangement, the forearm musculature of chick wing buds was analysed following grafts of the polarizing region or application of retinoic acid. Muscle patterns appear symmetrical with 'posterior' muscles now forming in the anterior part of the wing. When the number of muscles that develop is reduced, pattern symmetry is maintained, with loss of anterior muscles in the mid-line, especially dorsally. Strict anteroposterior ordering of muscles in duplicated patterns does not always occur. The number of muscles that develops bears some relationship to the number of forearm elements. Each muscle has a characteristic pattern of fast and slow fibres. In duplicated wings, each pair of symmetrically arranged muscles has the same fibre type pattern. Not only are proportions of fast and slow fibres similar, but local variations in fibre type arrangement within the muscle are also reproduced. This suggests that the cellular pattern within the new 'posterior' muscles at the anterior of the limb has been re-specified. In manipulated limb buds, which will develop a duplicated muscle pattern, there are no detectable changes in distribution and number of potentially myogenic cells, and fibre type patterning within early muscle masses also appears normal. In contrast, the splitting process that divides up muscle masses is altered. The appropriate fibre type arrangement only emerges after splitting is complete. This suggests that tissue patterning and cellular patterning occur at different times during muscle development.

Experimental analysis of the control of expression of the homeobox-gene Msx-1 in the developing limb and face.

Mouse mesenchyme was grafted into chick embryos to investigate the control of mesenchymal expression of Msx-1 in the developing limb and face. In situ hybridization, using species-specific probes, allows a comparison between Msx-1 expression in the graft and the host tissue. The results show that Msx-1 expression in both limb-to-limb and face-to-face grafts corresponds closely with the level of Msx-1 expression in the surrounding chick mesenchyme. Cells in grafts that end up within the host domain of Msx-1 express the gene irrespective of whether they were from normally expressing, or non-expressing, regions. Therefore Msx-1 expression in both the developing limb and the developing face appears to be position-dependent. Mesenchyme from each of the three major facial primordia behaved in the same way when grafted to the chick maxillary primordium. Reciprocal grafts between face and limb gave a different result: Msx-1 expression was activated when facial mesenchyme was grafted to the limb but not when limb mesenchyme was grafted to the face. This suggests either that there are quantitative or qualitative differences in two local signalling systems or that additional factors determine the responsiveness of the mesenchyme cells.

Cellular patterning of fast and slow fibres in the intermandibularis muscle of chick embryos.

The way in which the pattern of cell types arises during development of individual muscles was explored. The pattern of cellular differentiation resulting from the synthesis of particular fast and slow myosin heavy chains (MyHC) was investigated in the intermandibularis muscle in the lower jaw of chick embryos. The intermandibularis muscle has a proximodistal pattern of fibre type distribution. The distal region of the muscle contains a ratio of 1.5:1 fast to slow muscle fibres, which increases to > 2.5:1 in the proximal region. The intermandibularis muscle is assembled in a proximodistal sequence, with both fast and slow muscle cells differentiating within the earliest muscle and then establishing the specific pattern of cell types. This pattern is not dependent on a specific innervation source, as normal lower jaw muscles develop and the intermandibularis has the same graded cellular pattern when the mandibular primordium is grafted to the limb bud stump. Micromass cultures were used to explore the pool of potentially myogenic cells that are available to construct the muscles. Even before the muscle differentiates in vivo, both fast and slow cells are present in the primordia. These potentially myogenic cells are already distributed within the primordium in a proximodistal fashion that mimics the cellular pattern found in the muscle that develops.

A 15p+ variant shown to be a t(Y;15) with fluorescence in situ hybridisation.

Prenatal diagnosis in two successive pregnancies revealed the karyotype 46,XX,15p+ (pat). Using the Y heterochromatic probe pHY3.4 and fluorescence in situ hybridisation, the variant 15 was identified as a t(Y;15)(q12;p11). Interphase scanning alone would have given a false result in both prenatal assessments and in the phenotypically normal father.

Thixotropy in frog single muscle fibres.

The thixotropic properties of single muscle fibres have been investigated. The effect is larger than in whole muscle and although the time course of stiffness recovery is generally similar, there is an indication that two phases may be involved. Rigor, induced chemically, eliminates thixotropic behaviour.

Thixotropy: the effect of stimulation in frog muscle.

Recent reports have shown that the stiffness of relaxed frog muscle is not a fixed property, but is dependent on the previous history of movement (Lakie & Robson, 1988a). Passive movement decreases stiffness; when the muscle is subsequently allowed to rest it returns at a progressively declining rate to a level of stiffness that is close to its original value (Lakie & Robson, 1988b). We now report that the stiffness of relaxed muscle is also affected by prior tetanic electrical stimulation under isometric conditions. The effect of electrical stimulation is similar to passive movement in that both produce a temporary decrease in stiffness.

Thixotropic changes in human muscle stiffness and the effects of fatigue.

Two methods have been used to study the stiffness of the relaxed finger musculature after movement followed by various times at rest. The muscles stiffen considerably as the time at rest increases. The time course of this change has been plotted; it continues at a declining rate for at least 30 min. The increased stiffness after resting can be immediately reduced by active or passive movements but not by isometric efforts. These changes characterize a thixotropic system and suggest a long-term molecular rearrangement in relaxed muscle. Extensive eccentric exercise of the muscles under investigation reduces the overall stiffness and there is a concomitant increase in tremor on movement.

Thixotropy: stiffness recovery rate in relaxed frog muscle.

The stiffness of relaxed frog muscle is affected by its previous history of movement. The extent of this thixotropic effect is dependent on the size of the applied force (Lakie & Robson, 1988). The stiffness recovery following movement has now been studied at two temperatures, and has been shown to proceed at a logarithmically declining rate.

Thixotropy: the effect of stretch size in relaxed frog muscle.

Small forces were applied to isolated frog muscle; the resulting displacements were used to calculate muscle stiffness (elastic modulus, E). Stiffness is much greater for small forces within the range of the Short Range Elastic Component (SREC; Hill, 1968). 'Stirring' the muscle greatly reduces the stiffness, but only when the applied force is small. Stiffness subsequently returns to its original level. This strongly suggests that muscle thixotropy (Lakie, Walsh & Wright, 1984) is a property of the SREC.