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  • The use of PRV-Bartha to define premotor inputs to lumbar motoneurons in the neonatal spinal cord of the mouse. 20668534

    The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24-32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32-40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8-12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.The study establishes the time window for virally-labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks.
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  • The use of poly(N-[2-hydroxypropyl]-methacrylamide) hydrogel to repair a T10 spinal cord hemisection in rat: a behavioural, electrophysiological and anatomical examinatio ... 23614684

    There have been considerable interests in attempting to reverse the deficit because of an SCI (spinal cord injury) by restoring neural pathways through the lesion and by rebuilding the tissue network. In order to provide an appropriate micro-environment for regrowing axotomized neurons and proliferating and migrating cells, we have implanted a small block of pHPMA [poly N-(2-hydroxypropyl)-methacrylamide] hydrogel into the hemisected T10 rat spinal cord. Locomotor activity was evaluated once a week during 14 weeks with the BBB rating scale in an open field. At the 14th week after SCI, the reflexivity of the sub-lesional region was measured. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and increase the respiratory rate. Spinal cords were then collected, fixed and stained with anti-ED-1 and anti-NF-H antibodies and FluoroMyelin. We show in this study that hydrogel-implanted animals exhibit: (i) an improved locomotor BBB score, (ii) an improved breathing adjustment to electrically evoked isometric contractions and (iii) an H-reflex recovery close to control animals. Qualitative histological results put in evidence higher accumulation of ED-1 positive cells (macrophages/monocytes) at the lesion border, a large number of NF-H positive axons penetrating the applied matrix, and myelin preservation both rostrally and caudally to the lesion. Our data confirm that pHPMA hydrogel is a potent biomaterial that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses after SCI.
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  • The use of dentin matrix scaffold and dental follicle cells for dentin regeneration. 19767098

    Scaffold and inductive microenvironment are the two most important factors for dentin regeneration. They have been addressed with hydroxyapatite, tricalcium phosphate, polyglycolic acid, calcined bovine bone, and collagen, among other things. However, as of yet, no scaffold and inductive microenvironment combination has been shown to contribute to the regeneration of complete and prefabricated-shaped dentin tissues that include dentin, predentin and odontoblasts. To test the supporting and inductive effects of treated dentin matrix (TDM) on complete and prefabricated-shaped dentin regeneration, dental follicle cells (DFCs) were seeded onto TDM and further incubated for 1 and 2 weeks in vitro and for 2 and 4 weeks in vivo. The results show that in vitro, in addition to dentin sialoprotein (DSP) and dentin matrix protein 1 (DMP1) (regarded as identifying markers of odontoblasts), DFCs induced by TDM expressed osteocalcin, bone sialoprotein, type I collagen, osteopontin, osteonectin and alkaline phosphatase (all expressed by odontoblasts), and that complete and prefabricated-shaped dentin was successfully regenerated. Most importantly, it was found that in vivo TDM supports and induces regeneration of complete and prefabricated-shaped dentin, and regenerated dentin expresses DSP and DMP1, which are identifying dentin markers. Taken together, these results suggest that, for dentin regeneration, TDM is a suitable scaffold and inductive microenvironment and DFCs are a suitable cell type. The combination of TDM and DFCs may constitute a promising approach for future clinical dentin regeneration.
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    AP192F
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    Donkey Anti-Mouse IgG Antibody, FITC conjugate, Species Adsorbed

  • The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl s ... 1653021

    The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.
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  • The use of an in vitro 3D melanoma model to predict in vivo plasmid transfection using electroporation. 22244695

    A large-scale in vitro 3D tumor model was generated to evaluate gene delivery procedures in vivo. This 3D tumor model consists of a "tissue-like" spheroid that provides a micro-environment supportive of melanoma proliferation, allowing cells to behave similarly to cells in vivo. This functional spheroid measures approximately 1 cm in diameter and can be used to effectively evaluate plasmid transfection when testing various electroporation (EP) electrode applicators. In this study, we identified EP conditions that efficiently transfect green fluorescent protein (GFP) and interleukin 15 (IL-15) plasmids into tumor cells residing in the 3D construct. We found that plasmids delivered using a 6-plate electrode applying 6 pulses with nominal electric field strength of 500 V/cm and pulse-length of 20 ms produced significant increase of GFP (7.3-fold) and IL-15 (3.0-fold) expression compared to controls. This in vitro 3D model demonstrates the predictability of cellular response toward delivery techniques, limits the numbers of animals employed for transfection studies, and may facilitate future developments of clinical trials for cancer therapies in vivo.
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