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Sökning: WFRF:(Terenghi Giorgio)

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1.
  • Armstrong, Stephanie J., et al. (författare)
  • ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth
  • 2007
  • Ingår i: Tissue engineering. - : Mary Ann Liebert. - 1076-3279 .- 1557-8690. ; 13:12, s. 2863-2870
  • Tidskriftsartikel (refereegranskat)abstract
    • Tissue engineering using a combination of biomaterials and cells represents a new approach to nerve repair. We have investigated the effect that extracellular matrix (ECM) molecules have on Schwann cell (SC) attachment and proliferation on the nerve conduit material poly-3-hydroxybutyrate (PHB), and SC influence on neurite outgrowth in vitro. Initial SC attachment to PHB mats was unaffected by ECM molecules but proliferation increased (laminin > fibronectin > collagen). SCs seeded onto ECM-coated culture inserts suspended above a monolayer of NG108-15 cells determined the effect of released diffusible factors. The effect of direct contact between the two cell types on ECM molecules was also investigated. In both systems SCs enhanced neurite number per cell and percentage of NG108-15 cells sprouting neurites. NG108-15 cells grown in direct contact with SCs had significantly longer neurites than those exposed to diffusible factors when seeded on laminin or fibronectin. Diffusible factors released from SCs cultured on ECM molecules appear to initiate neurite outgrowth, whereas SC-neuron contact promotes neurite elongation. SC proliferation was maximal on poly-D-lysine-coated surfaces, but these cells did not influence neurite outgrowth to the levels of laminin or fibronectin. This suggests that ECM molecules enhance cell number and activate SCs to release neurite promoting factors. Addition of ECM molecules to PHB nerve conduits containing SCs is likely to provide benefits for the treatment of nerve injuries.
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2.
  • Armstrong, Stephanie J, et al. (författare)
  • Laminin activates NF-kappaB in Schwann cells to enhance neurite outgrowth.
  • 2008
  • Ingår i: Neuroscience Letters. - : Elsevier BV. - 0304-3940 .- 1872-7972. ; 439:1, s. 42-6
  • Tidskriftsartikel (refereegranskat)abstract
    • Extracellular matrix (ECM) molecules and Schwann cells (SCs) are important components of peripheral nerve regeneration. In this study, the role of the transcription factor nuclear factor kappa B (NF-kappaB) in SC activation in response to laminin and the subsequent effect on in vitro neurite outgrowth was investigated. Immunocytochemistry and Western blot analysis showed that compared with poly-d-lysine (PDL), laminin enhanced the phosphorylation of IkappaB and p65 NF-kappaB signalling proteins in SCs. Phospho NF-kappaB-p65 was localised to the nucleus indicating activation of NF-kappaB. To assess the functional effect of NF-kappaB activation, SCs plated on PDL or laminin were pre-treated with NF-kappaB inhibitors, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) or Z-leu-leu-leu-CHO (MG-132) before NG108-15 neuronal cells were seeded on the SC monolayer. After 24h co-culture in the absence of inhibitors, SCs seeded on laminin enhanced the mean number and length of neurites extended by NG108-15 cells (1.87+/-0.13 neurites; 238.74+/-8.53microm) compared with those cultured in the presence of SCs and PDL (1.26+/-0.07 neurites; 157.57+/-9.80microm). At 72h, neurite length had further increased to 321.83+/-6.60microm in the presence of SCs and laminin. Inhibition of NF-kappaB completely abolished the effect of laminin on SC evoked neurite outgrowth at 24h and reduced the enhancement of neurite length by over 60% at 72h. SC proliferation was unaffected by NF-kappaB inhibition suggesting that the NF-kappaB signalling pathway plays a discrete role in the activation of SCs and their neurotrophic potential.
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3.
  • Brohlin, Maria, et al. (författare)
  • Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells
  • 2009
  • Ingår i: Neuroscience research. - : Elsevier BV. - 0168-0102 .- 1872-8111. ; 64:1, s. 41-49
  • Tidskriftsartikel (refereegranskat)abstract
    • Cell-based therapies provide a clinically applicable and available alternative to nerve autografts. Our previous studies have characterised rat-derived mesenchymal stem cells (MSC) and here we have investigated the phenotypic, molecular and functional characteristics of human-derived MSC (hMSC) differentiated along a Schwann cell lineage. The hMSC were isolated from healthy human donors and the identity of the undifferentiated hMSC was confirmed by the detection of MSC specific cells surface markers. The hMSC were differentiated along a glial cell lineage using an established cocktail of growth factors including glial growth factor-2. Following differentiation, the hMSC expressed the key Schwann cell (SC) markers at both the transcriptional and translational level. More importantly, we show the functional effect of hMSC on neurite outgrowth using an in vitro co-culture model system with rat-derived primary sensory neurons. The number of DRG sprouting neurites was significantly enhanced in the presence of differentiated hMSC; neurite length and density (branching) were also increased. These results provide evidence that hMSC can undergo molecular, morphological and functional changes to adopt a SC-like behaviour and, therefore, could be suitable as SC substitutes for nerve repair in clinical applications.
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4.
  • Caddick, Jenny, et al. (författare)
  • Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage.
  • 2006
  • Ingår i: Glia. - : Wiley. - 0894-1491 .- 1098-1136. ; 54:8, s. 840-849
  • Tidskriftsartikel (refereegranskat)abstract
    • We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.
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5.
  • di Summa, Pietro G, et al. (författare)
  • Adipose-derived stem cells enhance peripheral nerve regeneration
  • 2010
  • Ingår i: Journal of plastic, reconstructive and aesthetic surgery. - : Elsevier. - 1878-0539 .- 1748-6815. ; 63:9, s. 1544-1552
  • Tidskriftsartikel (refereegranskat)abstract
    • Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.
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6.
  • di Summa, Pietro G, et al. (författare)
  • Extracellular matrix molecules enhance the neurotrophic effect of Schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions
  • 2013
  • Ingår i: Tissue Engineering. Part A. - : Mary Ann Liebert Inc. - 1937-3341 .- 1937-335X. ; 19:3-4, s. 368-379
  • Tidskriftsartikel (refereegranskat)abstract
    • Since the first reports of induction of adipose-derived stem cells (ASC) into neuronal and glial cell phenotypes, expectations have increased regarding their use in tissue engineering applications for nerve repair. Cell adhesion to extracellular matrix (ECM) is a basic feature of survival, differentiation, and migration of Schwann cells (SC) during nerve regeneration, and fibronectin and laminin are two key molecules of this process. Interaction between ECM and SC-like differentiated ASC (dASC) could potentially improve the neurotrophic potential of the stem cells. We have investigated the effect of ECM molecules on SC-like dASC in terms of proliferation, adhesion, and cell viability. Fibronectin and laminin did not affect the proliferation of dASC when compared with cell adherent tissue culture plastic, but significantly improved viability and cell attachment when dASC were exposed to apoptotic conditions. To assess the influence of the ECM molecules on dASC neurotrophic activity, dASC were seeded onto ECM-coated culture inserts suspended above dorsal root ganglia (DRG) sensory neurons. Neurite outgrowth of DRG neurons was enhanced when dASC were seeded on fibronectin and laminin when compared with controls. When DRG neurons and dASC were in direct contact on the various surfaces there was significantly enhanced neurite outgrowth and coculture with laminin-conditioned dASC produced the longest neurites. Compared with primary SCs, dASC grown on laminin produced similar levels of neurite outgrowth in the culture insert experiments but neurite length was shorter in the direct contact groups. Anti beta 1 integrin blocking antibody could inhibit baseline and dASC evoked neurite elongation but had no effect on outgrowth mediated by laminin-conditioned dASC. ECM molecules had no effect on the levels of nerve growth factor and brain-derived neurotrophic factor secretion from dASC. The results of the study suggest that ECM molecules can significantly improve the potential of dASC for nerve regeneration.
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7.
  • di Summa, Pietro G, et al. (författare)
  • Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts
  • 2011
  • Ingår i: Neuroscience. - : Elsevier BV. - 0306-4522 .- 1873-7544. ; 181:5, s. 278-291
  • Tidskriftsartikel (refereegranskat)abstract
    • Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.
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8.
  • Englezou, Pavlos C., et al. (författare)
  • Mitochondrial involvement in sensory neuronal cell death and survival
  • 2012
  • Ingår i: Experimental Brain Research. - : Springer Science and Business Media LLC. - 0014-4819 .- 1432-1106. ; 221:4, s. 357-367
  • Tidskriftsartikel (refereegranskat)abstract
    • Peripheral nerve injuries (PNI) are continuing to be an ever-growing socio-economic burden affecting mainly the young working population and the current clinical treatments to PNI provide a poor clinical outcome involving significant loss of sensation. Thus, our understanding of the underlying factors responsible for the extensive loss of the sensory cutaneous subpopulation in the dorsal root ganglia (DRG) that occurs following injury needs to be improved. The current investigations focus in identifying visual cues of mitochondria-related apoptotic events in the various subpopulations of sensory cutaneous neurons. Sensory neuronal subpopulations were identified using FastBlue retrograde labelling following axotomy. Specialised fluorogenic probes, MitoTracker Red and MitoTracker Orange, were employed to visualise the dynamic changes of the mitochondrial population of neurons. The results reveal a fragmented mitochondrial network in sural neurons following apoptosis, whereas a fused elongated mitochondrial population is present in sensory proprioceptive muscle neurons following tibial axotomy. We also demonstrate the neuroprotective properties of NAC and ALCAR therapy in vitro. The dynamic mitochondrial network breaks down following oxidative exposure to hydrogen peroxide (H2O2), but reinitiates fusion after NAC and ALCAR therapy. In conclusion, this study provides both qualitative and quantitative evidence of the susceptibility of sensory cutaneous sub-population in apoptosis and of the neuroprotective effects of NAC and ALCAR treatment on H2O2-challenged neurons.
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9.
  • Erba, Paolo, et al. (författare)
  • Neural differentiation and therapeutic potential of adipose tissue derived stem cells
  • 2010
  • Ingår i: Current stem cell research & therapy. - : Bentham Science Publishers Ltd.. - 1574-888X. ; 5:2, s. 153-160
  • Forskningsöversikt (refereegranskat)abstract
    • Neural tissue has historically been regarded as having poor regenerative capacity but recent advances in the growing fields of tissue engineering and regenerative medicine have opened new hopes for the treatment of nerve injuries and neurodegenerative disorders. Adipose tissue has been shown to contain a large quantity of adult stem cells (ASC). These cells can be easily harvested with low associated morbidity and because of their potential to differentiate into multiple cell types, their use has been suggested for a wide variety of therapeutic applications. In this review we examine the evidence indicating that ASC can stimulate nerve regeneration by both undergoing neural differentiation and through the release of a range of growth factors. We also discuss some of the issues that need to be addressed before ASC can be developed as an effective cellular therapy for the treatment of neural tissue disorders.
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10.
  • Erba, P, et al. (författare)
  • Regeneration potential and survival of transplanted undifferentiated adipose tissue-derived stem cells in peripheral nerve conduits
  • 2010
  • Ingår i: Journal of plastic, reconstructive & aesthetic surgery : JPRAS. - : Elsevier BV. - 1878-0539 .- 1748-6815. ; 63:12, s. e811-e817
  • Tidskriftsartikel (refereegranskat)abstract
    • Adipose tissue-derived stem cells (ADSCs) have shown potential for the treatment of nerve injuries. Most previous efforts have aimed at stimulating regeneration by using neural-differentiation protocols, but the potential of undifferentiated ADSCs to enhance axonal growth as well as their ability to transdifferentiate in situ have been poorly investigated. In this study, using a rat sciatic nerve model we show that ADSCs, transplanted in an artificial nerve conduit, stimulate axonal outgrowth from the proximal nerve stump and evoke greater Schwann cell (SC) proliferation/intrusion in the distal stump. To track the fate of the transplanted cells, we used green fluorescent protein (GFP)-labelling and polymerase chain reaction (PCR) for the detection of the sex determining region Y (SRY) gene in the donor male cells. Both methods indicated a lack of significant quantities of viable cells 14 days after transplantation. These results suggest that any regenerative effect of transplanted ADSCs is more likely to be mediated by an initial boost of released growth factors and/or by an indirect effect on endogenous SCs activity. Future studies need to address long-term cell survival in tissue-engineered nerve conduits to improve the neuroregenerative potential of ADSCs.
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11.
  • Hart, Andrew McKay, et al. (författare)
  • Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit
  • 2003
  • Ingår i: British Journal of Plastic Surgery. - : Elsevier. - 0007-1226 .- 1465-3087. ; 56:5, s. 444-450
  • Tidskriftsartikel (refereegranskat)abstract
    • The clinical outcome of peripheral nerve injuries remains disappointing, even in the ideal situation of a primary repair performed with optimal microsurgical techniques. Primary repair is appropriate for only about 85% of injuries, and outcome is worse following secondarynerverepair, partly owing to the reduced regenerative potential of chronically axotomised neurons. Leukaemiainhibitoryfactor (LIF) is a gp-130 neurocytokine that is thought to act as an ‘injury factor’, triggering the early-injury phenotype within neurons and potentially boosting their regenerative potential aftersecondarynerverepair. At 2–4 months after sciatic nerve axotomy in the rat, 1 cm gaps were repaired using either nerve isografts or poly-3-hydroxybutyrate conduits containing a calcium alginate and fibronectin hydrogel.Regeneration was determined by quantitative immunohistochemistry 6 weeks afterrepair, and the effect of incorporating recombinant LIF (100 ng/ml) into the conduits was assessed. LIF increased the regeneration distance in repairs performed after both 2 months (69%, P=0.019) and 4 months (123%, P=0.021), and was statistically comparable to nerve graft. The total area of axonal immunostaining increased by 21% (P>0.05) and 63% (P>0.05), respectively. Percentage immunostaining area was not increased in the 2 months group, but increased by 93% in the repairs performed 4 months after axotomy. Exogenous LIF, therefore, has a potential role in promoting peripheral nerveregenerationaftersecondaryrepair, and can be effectively delivered within poly-3-hydroxybutyrate bioartificialconduits used for nerverepair.
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12.
  • Hart, Andrew M, et al. (författare)
  • Frozen-section fluorescence microscopy and stereology in the quanti cation of neuronal death within dorsal root ganglia
  • 2004
  • Ingår i: Journal of Molecular Histology. - Dordrecht : Kluwer Academic Publishers. - 1567-2379 .- 1567-2387. ; 35:6, s. 565-580
  • Tidskriftsartikel (refereegranskat)abstract
    • Histochemical and morphological research increasingly relies upon quanti cation of complex biological systems. For such investigations to be meaningful, quanti cation techniques must meet the seemingly conflicting requirements of being theoretically robust, yet sufficiently practical to facilitate widespread applicability. Validity ought to be enhanced by theoretical simplicity, use of measured rather than assumed variables, and minimising observer interpretation. Practicality is facilitated by simplifying and reducing measurements, broadening applicability, and reducing costs and analysis time. As a result, quanti cation systems that rely upon sampling and estimation have been favoured over serial reconstruction techniques. To provide reliable estimates, sampling must be valid at all levels from tissue harvest, to the selection of microscope fields in which quanti cation is performed by techniques that account for the anisotropic distribution, and variable size of many elements in biological systems. These principles are embodied in the development of a stereological approach to the quanti cation of neuronal death within dorsal root ganglia after peripheral nerve injury. This frozen section technique is efficient and flexible, since it permits simultaneous morphological examination, TUNEL, or standard fluorescence immunohistochemistry, broadening its applicability. Section shrinkage is minimal, and counting by optical disection has proved to be time-efficient and sufficiently reproducible to reliably detect losses in the order of 5%, with minimal inter-observer variation. As is discussed, stereology has not yet met with universal acceptance, but by balancing theoretical validity with practical applicability, it has proved an excellent approach to the investigation of neuronal death within dorsal root ganglia.
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13.
  • Hart, Andrew M, et al. (författare)
  • Neuronal death after peripheral nerve injury and experimental strategies for neuroprotection.
  • 2008
  • Ingår i: Neurological Research. - 0161-6412 .- 1743-1328. ; 30:10, s. 999-1011
  • Tidskriftsartikel (refereegranskat)abstract
    • OBJECTIVE: Despite considerable microsurgical innovation in peripheral nerve repair, the outcome has improved little since the 1940s, reflecting surgical inability to adequately address the complex neurobiology of nerve injury and regeneration. Axotomy-induced neuronal death is potentially the most fundamental problem, and given recently published data, a review is timely. METHODS: Initial review of relevant doctoral theses from the University of Umeå, and Blond-McIndoe Research Laboratories, the University of Manchester, plus initial PubMed search including terms 'neuron death' and 'neuroprotection', subsequently expanded to relevant quoted articles. RESULTS: Various factors related to patient (principally age) and injury (Sunderland grade, proximity to cell body and mechanism) determine the extent of neuronal death, the mechanism of which is reviewed. A considerable proportion of sensory neurons (particularly small cutaneous afferents) die after distal injury and death is more widespread after proximal injury. Motor neurons are susceptible to post-ganglionic plexus and spinal root level injury. Root avulsion causes the greatest cell death. The time course of neuronal death is fortuitously slow and mainly occurs by a process akin to apoptosis. A therapeutic window therefore exists, as do potential neuroprotective targets. Nerve repair is partly neuroprotective, but must be performed early. Exogenous neurotrophic factor administration (e.g. in tissue engineered conduits) is beneficial, but not practical for various reasons. In contrast, adjuvant neuroprotective pharmacotherapy is practical, and two clinically safe agents are reviewed. Acetyl-L-carnitine arrests sensory neuronal death and speeds up regeneration. N-acetyl-cysteine provides comparable sensory neuronal protection via mitochondrial preservation and protects motor neurons. Both agents are well characterized experimentally and highly effective even after clinically relevant delays between injury and treatment. Barriers to translational research are being addressed. DISCUSSION: The future of peripheral nerve repair lies in modulating neurobiology at the time of injury, repair and during regeneration. Neuroprotection may be an essential component of that therapeutic package.
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14.
  • Hart, Andrew McKay, et al. (författare)
  • Pharmacological enhancement of peripheral nerve regeneration in the rat by systemic acetyl-L-carnitine treatment
  • 2002
  • Ingår i: Neuroscience Letters. - : Elsevier. - 0304-3940 .- 1872-7972. ; 334:3, s. 181-185
  • Tidskriftsartikel (refereegranskat)abstract
    • Peripheral nerve trauma remains a major cause of morbidity, largely due to the death of similar to40% of innervating sensory neurons, and to slow regeneration after repair. Acetyl-L-carnitine (ALCAR) is a physiological peptide that virtually eliminates sensory neuronal death, and may improve regeneration after primary nerve repair. This study determines the effect of ALCAR upon regeneration after secondary nerve repair, thereby isolating its effect upon neuronal regenerative capacity. Two months after unilateral sciatic nerve division 1 cm nerve graft repairs were performed (n = 5), and treatment with 50 mg/kg/day ALCAR was commenced for 6 weeks until harvest. Regeneration area and distance were determined by quantitative immunohistochemistry. ALCAR treatment significant increased immunostaining for both nerve fibres (total area 264% increase, P < 0.001; percentage area 229% increase, P < 0.001), and Schwann cells (total area 264% increase, P < 0.05; percentage area 86% increase, P < 0.05), when compared to no treatment. Regeneration into the distal stump was greatly enhanced (total area 2242% increase, P = 0.008; percentage area 3034% increase, P = 0.008). ALCAR significantly enhances the regenerative capacity of neurons that survive peripheral nerve trauma, in addition to its known neuroprotective effects.
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15.
  • Hart, Andrew McKay, et al. (författare)
  • Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat : timecourse of cell death & elimination
  • 2002
  • Ingår i: Experimental Brain Research. - : Springer-Verlag New York. - 0014-4819 .- 1432-1106. ; 142:3, s. 308-318
  • Tidskriftsartikel (refereegranskat)abstract
    • The timecourse of cell death in adult dorsal root ganglia after peripheral axotomy has not been fully characterised. It is not clear whether neuronal death begins within I week of axotomy or continues beyond 2 months after axotomy. Similarly, neither the timecourse of satellite cell death in the adult, nor the effect of nerve repair has been described. L4 and L5 dorsal root ganglia were harvested at 1-14 days, 1-6 months after sciatic nerve division in the adult rat, in accordance with the Animals (Scientific Procedures) Act 1986. In separate groups the nerve was repaired either immediately or following a 1-week delay, and the ganglia were harvested 2 weeks after the initial transection. Microwave permeabilisation and triple staining enabled combined TUNEL staining, morphological examination and neuron counting by the stereological optical dissector technique. TUNEL-positive neurons, exhibiting a range of morphologies, were seen at all timepoints (peak 25 cells/group 2 weeks after axotomy) in axotomised ganglia only. TUNEL-positive satellite cell numbers peaked 2 months after axotomy and were more numerous in axotomised than control ganglia. L4 control ganglia contained 13,983 (SD 568) neurons and L5, 16,285 (SD 1,313). Neuron loss was greater in L5 than L4 axotomised ganglia, began at I week (15%, P=0.045) post-axotomy, reached 35% at 2 months (P<0.001) and was not significantly greater at 4 months or 6 months. Volume of axotomised ganglia fell to 19% of control by 6 months (P<0.001). In animals that underwent nerve repair, both the number of TUNEL-positive neurons and neuron loss were reduced. Immediate repair was more protective than repair after a 1-week delay. Thus TUNEL positivity precedes actual neuron loss, reflecting the time taken to complete cell death and elimination. Neuronal death begins within I day of peripheral axotomy, the majority occurs within the first 2 months, and limited death is still occurring at 6 months. Neuronal death is modulated by peripheral nerve repair and by its timing after axotomy. Secondary satellite cell death also occurs, peaking 2 months after axotomy. These results provide a logical framework for future research into neuronal and satellite cell death within the dorsal root ganglia and provide further insight into the process of axotomy induced neuronal death.
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16.
  • Hart, Andrew McKay, et al. (författare)
  • Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.
  • 2004
  • Ingår i: Neuroscience. - : Elsevier BV. - 0306-4522 .- 1873-7544. ; 125:1, s. 91-101
  • Tidskriftsartikel (refereegranskat)abstract
    • Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.
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17.
  • Hart, Andrew McKay, et al. (författare)
  • Systemic acetyl-L-carnitine eliminates sensory neuronal loss after peripheral axotomy : a new clinical approach in the management of peripheral nerve trauma
  • 2002
  • Ingår i: Experimental Brain Research. - : Springer-Verlag New York. - 0014-4819 .- 1432-1106. ; 145:2, s. 182-189
  • Tidskriftsartikel (refereegranskat)abstract
    • Several hundred thousand peripheral nerve injuries occur each year in Europe alone. Largely due to the death of around 40% of primary sensory neurons, sensory outcome remains disappointingly poor despite considerable advances in surgical technique; yet no clinical therapies currently exist to prevent this neuronal death. Acetyl-L-carnitine (ALCAR) is a physiological peptide with roles in mitochondrial bioenergetic function, which may also increase binding of nerve growth factor by sensory neurons. Following unilateral sciatic nerve transection, adult rats received either one of two doses of ALCAR or sham, or no treatment. Either 2 weeks or 2 months later, L4 and L5 dorsal root ganglia were harvested bilaterally, in accordance with the Animal (Scientific Procedures) Act 1986. Neuronal death was quantified with a combination of TUNEL [TdT (terminal deoxyribonucleotidyl transferase) uptake nick end labelling] and neuron counts obtained using the optical disector technique. Sham treatment had no effect upon neuronal death. ALCAR treatment caused a large reduction in the number of TUNEL-positive neurons 2 weeks after axotomy (sham treatment 33/group; low-dose ALCAR 6/group, P=0.132; high-dose ALCAR 3/group, P<0.05), and almost eliminated neuron loss (sham treatment 21%; low-dose ALCAR 0%, P=0.007; high-dose ALCAR 2%, P<0.013). Two months after axotomy the neuroprotective effect of high-dose ALCAR treatment was preserved for both TUNEL counts (no treatment five/group; high-dose ALCAR one/group) and neuron loss (no treatment 35%; high-dose ALCAR -4%, P<0.001). These results provide further evidence for the role of mitochondrial bioenergetic dysfunction in post-traumatic sensory neuronal death, and also suggest that acetyl-L-carnitine may be the first agent suitable for clinical use in the prevention of neuronal death after peripheral nerve trauma.
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18.
  • Hart, Andrew, et al. (författare)
  • Tissue Engineering for Peripheral Nerve Regeneration
  • 2011
  • Ingår i: Tissue Engineering. - Berlin : Springer Berlin/Heidelberg. - 9783642028236 - 9783642028243 ; , s. 245-262
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • The outcome of peripheral nerve repair has changed very little over the past 50 years, and clinical outcomes remain generally poor. Surgical technique has evolved to a high level of technical microsurgical proficiency, but this approach remains unable to adequately address the neurobiological barriers to the optimization of nerve regeneration. Reconstruction of complex segmental injuries, as in the brachial plexus, additionally requires a considerable length of interpositional nerve autograft, which may be unobtainable without considerable donor morbidity.Research has therefore turned to the modulation of the repair-site environment to optimise nerve regeneration across neurorraphies, and to the creation of nerve conduits to reduce the need for nerve autograft. Tissue engineering has involved the use of growth factors to modulate neuronal and glial cell behaviour, and the creation of macro, micro and nanoscale constructs from a variety of materials in order to replace the connective tissue functions of nerve autograft. Implantation of cultured Schwann and stem cells is an area of particular development given the necessity of viable support cells to facilitate neuronal growth. These approaches are reviewed in the light of current published work.The future of peripheral nerve repair lies in the modulation of neuronal and glial cell responses to nerve injury, and during regeneration, while taking account of the clinical requirements and practical limitations. The peripheral nervous system also provides a simpler model for nerve regeneration than the central nervous system, but with translational potential.
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19.
  • Kalbermatten, Daniel F, 1969-, et al. (författare)
  • Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit
  • 2008
  • Ingår i: Journal of plastic, reconstructive and aesthetic surgery. - : Elsevier BV. - 1878-0539 .- 1748-6815. ; 61:6, s. 669-675
  • Tidskriftsartikel (refereegranskat)abstract
    • Peripheral nerve injury presents with specific problems of neuronal reconstructions, and from a clinical viewpoint a tissue engineering approach would facilitate the process of repair and regeneration. We have previously used artificial nerve conduits made from bioresorbable poly-3-hydroxybutyrate (PHB) in order to refine the ways in which peripheral nerves are repaired and reconnected to the target muscles and skin. The addition of Schwann cells (SC) or differentiated mesenchymal stem cells (dMSC) to the conduits enhances regeneration. In this study, we have used a matrix based on fibrin (Tisseel) to fill optimally the nerve-conduits with cells. In vitro analysis showed that both SC and MSC adhered significantly better to PHB in the presence of fibrin and cells continued to maintain their differentiated state. Cells were more optimally distributed throughout the conduit when seeded in fibrin than by delivery in growth medium alone. Transplantation of the nerve conduits in vivo showed that cells in combination with fibrin matrix significantly increased nerve regeneration distance (using PGP9.5 and S100 distal and proximal immunohistochemistry) when compared with empty PHB conduits. This study shows the beneficial combinatory effect of an optimised matrix, cells and conduit material as a step towards bridging nerve gaps which should ultimately lead to improved functional recovery following nerve injury.
  •  
20.
  • Kalbermatten, Daniel F, 1969-, et al. (författare)
  • New fibrin conduit for peripheral nerve repair
  • 2009
  • Ingår i: Journal of reconstructive microsurgery. - : Georg Thieme Verlag KG. - 0743-684X .- 1098-8947. ; 25:1, s. 27-33
  • Tidskriftsartikel (refereegranskat)abstract
    • An ideal substitute to treat a nerve gap has not been found. Initially, silicone conduits were employed. Later, conduits were fabricated from collagen or polyesters carbonates. More recently, it has been shown that a bioresorbable material, poly-3-hydroxybutyrate (PHB), can enhance nerve repair. The present investigation shows the use of fibrin as a conduit to guide nerve regeneration and bridge nerve defects. In this study we prepared and investigated a novel nerve conduit made from fibrin glue. Using a rodent sciatic nerve injury model (10-mm gap), we compared the extent of nerve regeneration through the new fibrin conduits versus established PHB conduits. After 2 and 4 weeks, conduits containing proximal and distal stumps were harvested. We evaluated the initial axon and Schwann cell stimulation using immunohistochemistry. The conduits presented full tissue integration and were completely intact. Axons crossed the gap after 1 month. Immunohistochemistry using the axonal marker PGP 9.5 showed a superior nerve regeneration distance in the fibrin conduit compared with PHB (4.1 mm versus 1.9 mm). Schwann cell intrusion (S100 staining) was similarly enhanced in the fibrin conduits, both from the proximal (4.2 mm versus 2.1 mm) and distal ends (3.2 mm versus 1.7 mm). These findings suggest an advantage of the new fibrin conduit for the important initial phase of peripheral nerve regeneration. The use of fibrin glue as a conduit is a step toward a usable graft to bridge peripheral nerve lesions. This might be clinically interesting, given the widespread acceptance of fibrin glue among the surgical community.
  •  
21.
  • Kalbermatten, Daniel F, 1969-, et al. (författare)
  • Schwann cell strip for peripheral nerve repair
  • 2008
  • Ingår i: Journal of Hand Surgery - British and European Volume. - : SAGE Publications. - 0266-7681 .- 1532-2211. ; 33:5, s. 587-594
  • Tidskriftsartikel (refereegranskat)abstract
    • Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.
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22.
  • Kelly, Edward J., et al. (författare)
  • End-to-side nerve coaptation : a qualitative and quantitative assessment in the primate
  • 2007
  • Ingår i: Journal of Plastic, Reconstructive & Aesthetic Surgery. - : Elsevier. - 1748-6815 .- 1878-0539. ; 60:1, s. 1-12
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • There are several reasons why end-to-side nerve coaptation has not been widely adopted clinically. Among these are the putative damage inflicted on the donor nerve and the variable quality of the regeneration in the recipient nerve. So far experiments on end-to-side nerve repair have been short term and mostly carried out on rats. This long-term study of end-to-side nerve repair of ulnar to median and median to ulnar nerve was performed using adult nonhuman primates. Eleven nerve repairs were studied at different time points. Eighteen, 22, 33 and 57 months after surgery a qualitative and quantitative analysis of the donor nerve and regenerating nerve revealed variable levels of percentage axonal regeneration compared with matched controls (1.4%-136%). Morphological evidence of donor nerve damage was identified distal to the coaptation site in four of the 11 cases, and in these cases the best axonal regeneration in the corresponding recipient nerves was observed. This donor nerve damage could neither be demonstrated in terms of a decrease in axon counts distal to the coaptation nor as donor target organ denervation. Recipient target organ regeneration like the axonal regeneration varied, with evidence of motor regeneration in eight out of 11 cases and sensory regeneration, as measured by percentage innervation density compared with matched controls, varied from 12.5% to 49%. Results from the present study demonstrate that the end-to-side coaptation technique in the nonhuman primate does not give predictable results. In general the motor recovery appeared better than the sensory and in those cases where donor nerve damage was observed there was better motor and sensory regeneration overall than in the remaining cases.
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23.
  • Kelly, Edward J, et al. (författare)
  • Nerve fibre and sensory end organ density in the epidermis and papillary dermis of the human hand.
  • 2005
  • Ingår i: Br J Plast Surg. - : Elsevier BV. - 0007-1226. ; 58:6, s. 774-9
  • Tidskriftsartikel (refereegranskat)abstract
    • Quantification of sensory recovery after peripheral nerve surgery is difficult and no accurate techniques are available at present. Quantification of reinnervated skin has been used experimentally, and in some clinical studies, but the lack of knowledge about the normal sensory distribution has been a problem. The purpose of this study was, therefore, to map the density of sensory end organs, nerve fibres and free nerve endings in the glabrous skin of the human hand. Skin biopsies were taken from patients undergoing acute and elective hand surgery. Nerve fibres were stained in the epidermis and papillary dermis and quantified in five sites on the palm of the hand, using protein gene product 9.5 immunoreactivity-a panneuronal marker. The finger tip skin was found to have more than twice the nerve fibre density in the papillary dermis than the skin of the palm, and the number of Meissner corpuscles in the finger tip was also higher than in the palm. We found a reduction in innervation density with increasing age in the dermis, however, that was not the case for the epidermis. The innervation of the epidermis showed high interindividual variability and unlike the papillary dermis did not display any pattern of distribution in the hand.
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24.
  • Kingham, Paul J, et al. (författare)
  • Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro.
  • 2007
  • Ingår i: Exp Neurol. - : Elsevier BV. - 0014-4886. ; 207:2, s. 267-74
  • Tidskriftsartikel (refereegranskat)abstract
    • Experimentally, peripheral nerve repair can be enhanced by Schwann cell transplantation but the clinical application is limited by donor site morbidity and the inability to generate a sufficient number of cells quickly. We have investigated whether adult stem cells, isolated from adipose tissue, can be differentiated into functional Schwann cells. Rat visceral fat was enzymatically digested to yield rapidly proliferating fibroblast-like cells, a proportion of which expressed the mesenchymal stem cell marker, stro-1, and nestin, a neural progenitor protein. Cells treated with a mixture of glial growth factors (GGF-2, bFGF, PDGF and forskolin) adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and western blotting indicated that the treated cells expressed the glial markers, GFAP, S100 and p75, indicative of differentiation. When co-cultured with NG108-15 motor neuron-like cells, the differentiated stem cells enhanced the number of NG108-15 cells expressing neurites, the number of neurites per cell and the mean length of the longest neurite extended. Schwann cells evoked a similar response whilst undifferentiated stem cells had no effect. These results indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and may be of benefit for treatment of peripheral nerve injuries.
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25.
  • Kingham, Paul J, et al. (författare)
  • Notch independent signalling mediates Schwann cell-like differentiation of adipose derived stem cells
  • 2009
  • Ingår i: Neuroscience Letters. - : Elsevier. - 0304-3940 .- 1872-7972. ; 467:2, s. 164-168
  • Tidskriftsartikel (refereegranskat)abstract
    • Adipose derived stem cells (ASC) differentiate into a Schwann cell (SC)-like phenotype but the signalling pathways mediating this are unknown. We hypothesised that notch might be involved, given its important role in regulating SC development. Rat ASC were differentiated using bFGF, PDGF, GGF-2 and forskolin. RT-PCR analysis showed that mRNA for notch-1 and notch-2 receptors and the notch responsive gene, hes-1, were expressed throughout the differentiation process whereas jagged-1 a notch ligand, and the hey-1 gene were markedly down-regulated. In contrast delta-1 was up-regulated with differentiation and was strongly expressed by rat primary SC. Treatment of ASC with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor which blocks notch signalling, had no effect on up-regulation of SC proteins S100 or GFAP during differentiation. Furthermore, when co-cultured with NG108-15 neurons, differentiated ASC cultures treated in the absence or presence of DAPT enhanced neurite outgrowth to similar levels. Differentiated ASC expressed PMP-22 but P0 was only present when co-cultured with dorsal root ganglia neurons. DAPT did not affect the expression of these myelin proteins. Thus, ASC express components of the notch signalling pathway but our studies suggest notch is unlikely to play a role in the neurotrophic activity and myelination capability of ASC differentiated into SC-like cells.
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26.
  • Kingham, Paul J, et al. (författare)
  • Stem cell and neuron co-cultures for the study of nerve regeneration
  • 2011
  • Ingår i: 3D Cell Culture. - New York : Humana Press. - 9781607619833 - 9781607619840 ; , s. 115-127
  • Bokkapitel (refereegranskat)abstract
    • Many experimental in vivo studies have indicated that Schwann cells are key facilitators of peripheral nerve regeneration but their clinical therapeutic potential may be limited. Recent advances suggest that stem cell therapy could one day be used to treat nerve traumas. We have shown how adult stem cells can be differentiated into a Schwann cell phenotype, characterised by expression of glial cell proteins and promotion of neurite outgrowth. The development of new cell culture models which mimic the in vivo regeneration environment will help us to better understand the functional benefits of these cells. Here, we describe a stepwise approach towards this, moving from traditional two-dimensional non-contact co-cultures to new three-dimensional models utilising fibrin matrices.
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27.
  • Mantovani, Cristina, 1974-, et al. (författare)
  • Bone marrow- and adipose-derived stem cells show expression of myelin mRNAs and proteins
  • 2010
  • Ingår i: Regenerative medicine. - : Future Medicine Ltd. - 1746-076X .- 1746-0751. ; 5:3, s. 403-410
  • Tidskriftsartikel (refereegranskat)abstract
    • Aims: PNS myelin is formed by Schwann cells (SCs). In this study, we applied an in vitro model to study myelin formation, using bone marrow mesenchymal stem cells and adipose-derived stem cells differentiated into SC-like cells and co-cultured with dissociated adult dorsal root ganglia neurons. Methods: Immunocytochemistry, reverse transcription-PCR and western blotting techniques were used to investigate the expression of myelin proteins at both the transcriptional and translational level. Results: Transcripts for protein zero, peripheral myelin protein 22 and myelin basic protein were detected in differentiated stem cells following co-culture with neuronal cells. Furthermore, protein zero, peripheral myelin protein 22 and myelin basic proteins were recognized in the co-cultures. These results were consistent with immunostaining of myelin proteins and with observation by electron microscopy. Conclusion: Both types of adult stems cells differentiated into SC-like cells have potential to myelinate neuronal cells during regeneration, being functionally identical to SCs of the PNS.
  •  
28.
  • Mantovani, Cristina, et al. (författare)
  • Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages
  • 2012
  • Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 318:16, s. 2034-2048
  • Tidskriftsartikel (refereegranskat)abstract
    • Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker (R) staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.
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29.
  • Mantovani, Maria Cristina, 1974- (författare)
  • Schwann cells and mesenchymal stem cells as promoter of peripheral nerve regeneration
  • 2011
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The transplantation of primary Schwann cells (SC) has been shown to improve nerve regeneration. However, to monitor the survival of transplanted cells within the host, a stable labelling method is required. The in vitro characteristics of green fluorescent protein labelled SC (GFP SC) and their effects in an in vivo peripheral nerve injury model were investigated.   The GFP-SC were readily visualised ex vivo and stimulated significantly better axonal regeneration compared to controls. Clinical use of autologous SC for the treatment of nerve injuries is of limited use due to difficulty in obtaining clinically useful numbers. However, bone marrow mesenchymal stem cells (MSC) can trans-differentiate into SC like cells (dMSC). The in vitro and in vivo differentiation of MSC was explored, and the study extended to include the easily-accessible adipose stem cells (ASC).  In vitro, glial growth factor stimulated MSC express S100, a SC marker, and its expression is maintained following in vivo transplantation.  Similarly, untreated MSC transplanted in vivo also expressed S100, which indicates glial differentiation in response to local cytokines and growth factors. Using an in vitro model, comprising dMSC or dASC co-cultured with adult dorsal root ganglia (DRG) neurons, the capacity of the dMSC and SC like differentiated ASC (dASC) to promote axon myelination was verified: both cell types expressed transcripts for protein zero, peripheral myelin protein-22 and myelin basic protein.The potential of stem cells in nerve repair may be limited by innate cellular senescence or donor age affecting cell functionality thus it was essential to determine the effects of donor age on morphology and functionality of stem cells.  The proliferation rates, expression of senescence markers (p38 and p53) and the stimulation of neurite outgrowth from DRG neurons by stem cells isolated from neonatal, young or old rats were very similar. However, the distribution and ultrastructure of mitochondria in dMSC and dASC from young and old rats were quite different, and seem to indicate physiological senescence of the aged cells.  Given the wide-ranging influence of Notch signalling in cell differentiation, including the neural crest to a glial cell type switch, and self-renewal in mammals, its role in the differentiation of stem cells to SC was investigated. The mRNA for notch-1 and -2 receptors were expressed in the dASC, blockage of notch signaling did not affect the neurotrophic and myelination potential of dASC. In conclusion, these findings show that GFP labelling has no deleterious effect on SC survival and function. MSC and ASC differentiated into glial-type cells acquire SC morphology, and express characteristic SC markers, and the differentiation process was independent of the Notch signaling pathway. Also, following transplantation into a nerve gap injury dMSC improve regeneration. This study established that following co-culture with DRG neurons, dMSC and dASC were able to express peripheral myelin proteins.  Also, the functional bioactivity of these cells is independent of the donor animal age. Finally, although the glial lineage differentiated aged cells characterized in this study expressed markers typical of senescence they retained the potential to support axon regeneration.
  •  
30.
  • Mohanna, Pari-Naz, et al. (författare)
  • Composite PHB-GGF conduit for long nerve gap repair : a long-term evaluation
  • 2005
  • Ingår i: Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery. - : Informa UK Limited. - 0284-4311 .- 1651-2073. ; 39:3, s. 129-137
  • Tidskriftsartikel (refereegranskat)abstract
    • Two to four cm nerve gaps in the rabbit common peroneal nerve were bridged with poly-3-hydroxybutyrate (PHB) conduits containing either glial growth factor (GGF) (PHB-GGF) or alginate matrix (PHB-ALG), and with empty PHB conduit (E-PHB). PHB-GGF significantly increased nerve regeneration up to 63 days following repair of long nerve gaps and the regeneration was sustained long term leading to motor organ reinnervation. At 120 days postoperatively, GGF addition significantly increased the quantity of Schwann cell and axonal regeneration compared to those in control conduits. In PHB-GGF conduits there were more minifascicles of myelinated fibres compared to the controls. The distal nerve of PHB-GGF and E-PHB conduits showed greater regeneration than that of PHB-ALG grafts, although all distal nerves contained fewer myelinated fibres than grafted conduits. Consistently, PHB-GGF conduits significantly reduced the muscle mass percentage loss compared to controls. In conclusion, GGF-containing conduits promoted sustained axonal regeneration and improved target muscle reinnervation.
  •  
31.
  • Novikov, Lev N, et al. (författare)
  • A novel biodegradable implant for neuronal rescue and regeneration after spinal cord injury.
  • 2002
  • Ingår i: Biomaterials. - 0142-9612 .- 1878-5905. ; 23:16, s. 3369-76
  • Tidskriftsartikel (refereegranskat)abstract
    • After spinal cord injury, the severed neuronal pathways fail to regenerate spontaneously. This study describes a biodegradable implant using poly-beta-hydroxybutyrate (PHB) fibers as carrier scaffold for matrix components and cell lines supporting neuronal survival and regeneration after spinal cord injury. After cervical spinal cord injury in adult rats, a graft consisting of PHB fibers coated with alginate hydrogel + fibronectin was implanted in the lesion cavity. In control groups, PHB was omitted and only alginate hydrogel or fibronectin, or their combination, were used for grafting. In addition, comparisons were made with animals treated intrathecally after spinal cord injury with the neurotrophic factors BDNF or NT-3. The neurons of the rubrospinal tract served as experimental model. In untreated animals, 45% of the injured rubrospinal neurons were lost at 8 weeks postoperatively. Implantation of the PHB graft reduced this cell loss by 50%, a rescuing effect similar to that obtained after treatment with BDNF or NT-3. In the absence of PHB support, implants of only alginate hydrogel or fibronectin, or their combination, had no effect on neuronal survival. After addition of neonatal Schwann cells to the PHB graft, regenerating axons were seen to enter the graft from both ends and to extend along its entire length. These results show that implants using PHB as carrier scaffold and containing alginate hydrogel, fibronectin and Schwann cells can support neuronal survival and regeneration after spinal cord injury
  •  
32.
  • Novikova, Liudmila N, et al. (författare)
  • Alginate hydrogel and matrigel as potential cell carriers for neurotransplantation.
  • 2006
  • Ingår i: Journal of Biomedical Materials Research part A. - : Wiley. - 1549-3296 .- 1552-4965. ; 77:2, s. 242-252
  • Tidskriftsartikel (refereegranskat)abstract
    • Development of biosynthetic conduits carrying extracellular matrix molecules and cell lines expressing neurotrophic growth factors represents a novel and promising strategy for spinal cord and peripheral nerve repair. In the present in vitro study, the compatibility and growth-promoting effects of (i) alginate hydrogel, (ii) alginate hydrogel complemented with fibronectin, and (iii) matrigel were compared between olfactory ensheathing cells (OECs), Schwann cells (SCs), and bone marrow stromal cells (BMSCs). Neurite outgrowth from embryonic dorsal root ganglia (DRG) neurons was used to assess the efficacy of the hydrogels alone or in combination with cultured cells to promote axonal regeneration. The result showed that alginate hydrogel transformed OECs, SCs, and BMSCs into atypical cells with spherical shape and inhibited their metabolic activity. Combination of alginate hydrogel with fibronectin promoted only OECs proliferation. Alginate hydrogel also inhibited outgrowth of DRG neurites, although this effect was attenuated by addition of fibronectin, SCs, or BMSCs. In contrast, matrigel stimulated cell proliferation, preserved the typical morphological features of the cultured cells and induced massive sprouting of DRG neurites. Addition of cultured cells to matrigel did not further improve DRG neurite outgrowth. The present findings suggest that addition of extracellular matrix should be considered when engineering biosynthetic scaffolds on the basis of alginate hydrogels.
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33.
  • Ouasti, Sihem, et al. (författare)
  • The CD44/integrins interplay and the significance of receptor binding and re-presentation in the uptake of RGD-functionalized hyaluronic acid
  • 2012
  • Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 33:4, s. 1120-1134
  • Tidskriftsartikel (refereegranskat)abstract
    • We have studied the interplay between two endocytic receptors for a carrier structure bearing two complementary ligands. Hyaluronic acid (HA; three different molecular weights) was functionalized with an RGD-containing peptide; this ancillary ligand allows the macromolecule to bind to alpha(v) integrins in addition to the classical HA internalization receptor (CD44). The uptake of HA-RGD and of native HA was assessed in a phagocytic cell model (J774.2 murine macrophages), studying the kinetics of internalization and its mechanistic details. Indications of a synergic binding to integrins and CD44 emerged for HA-RGD; possibly, a first binding to integrins allows for a pre-concentration of the macromolecule on the cell surface, which is then followed by its binding to CD44. The endocytic mechanism and kinetics appeared then dominated by CD44, which has a much slower turnover than integrins. In this study we have demonstrated that the knowledge of the rate-determining steps of the internalization of a carrier is necessary for assessing its performance. In this case, the presence of multiple ligands on a carrier was beneficial in some respect (e.g. in improved binding/targeting), but may not be sufficient to overcome penetration barriers that arise from slow receptor re-presentation. (C) 2011 Elsevier Ltd. All rights reserved.
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34.
  • Reid, Adam J, et al. (författare)
  • Long term peripheral nerve regeneration using a novel PCL nerve conduit
  • 2013
  • Ingår i: Neuroscience Letters. - : Elsevier. - 0304-3940 .- 1872-7972. ; 544, s. 125-30
  • Tidskriftsartikel (refereegranskat)abstract
    • The gold standard in surgical management of a peripheral nerve gap is currently autologous nerve grafting. This confers patient morbidity and increases surgical time therefore innovative experimental strategies towards engineering a synthetic nerve conduit are welcome. We have developed a novel synthetic conduit made of poly ε-caprolactone (PCL) that has demonstrated promising peripheral nerve regeneration in short-term studies. This material has been engineered to permit translation into clinical practice and here we demonstrate that histological outcomes in a long-term in vivo experiment are comparable with that of autologous nerve grafting. A 1cm nerve gap in a rat sciatic nerve injury model was repaired with a PCL nerve conduit or an autologous nerve graft. At 18 weeks post surgical repair, there was a similar volume of regenerating axons within the nerve autograft and PCL conduit repair groups, and similar numbers of myelinated axons in the distal stump of both groups. Furthermore, there was evidence of comparable re-innervation of end organ muscle and skin with the only significant difference the lower wet weight of the muscle from the PCL conduit nerve repair group. This study stimulates further work on the potential use of this synthetic biodegradable PCL nerve conduit in a clinical setting.
  •  
35.
  • Reid, Adam J, et al. (författare)
  • N-acetylcysteine alters apoptotic gene expression in axotomised primary sensory afferent subpopulations.
  • 2009
  • Ingår i: Neuroscience research. - : Elsevier BV. - 0168-0102 .- 1872-8111. ; 65:2, s. 148-155
  • Tidskriftsartikel (refereegranskat)abstract
    • Novel approaches are required in peripheral nerve injury management because current surgical techniques, which do not address axotomy-induced neuronal death, lead to deficient sensory recovery. Sensory neuronal death has functional preference with cutaneous neurons dying in great numbers whilst muscle afferents survive axotomy. This offers the potential of comparing similar cell types that suffer distinct fates upon nerve injury. Here, a novel approach, combining in vivo rat nerve injury model with laser microdissection and quantitative real-time polymerase chain reaction, identifies crucial disparities in apoptotic gene expression attributable to subpopulations of differing sensory modalities and examines the response to N-acetylcysteine (NAC) therapy. We show that axotomised muscle afferent neurons survive injury due to a neuroprotective response which markedly downregulates Bax and caspase-3 mRNA. In contrast, axotomised cutaneous sensory neurons significantly upregulate caspase-3 and alter both Bcl-2 and Bax expression such that pro-apoptotic Bax predominates. N-Acetylcysteine (NAC) intervention promotes neuroprotection of cutaneous sensory neurons through considerable upregulation of Bcl-2 and downregulation of both Bax and caspase-3 mRNA. The data presented identifies differential activation of apoptotic genes in axotomised neuronal subpopulations. Furthermore, NAC therapy instigates apoptotic gene expression changes in axotomised neurons, thereby offering pharmacotherapeutic potential in the clinical treatment of nerve injury.
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36.
  • Reid, Adam J, et al. (författare)
  • Nerve repair with adipose-derived stem cells protects dorsal root ganglia neurons from apoptosis
  • 2011
  • Ingår i: Neuroscience. - : Elsevier BV. - 0306-4522 .- 1873-7544. ; 199, s. 515-522
  • Tidskriftsartikel (refereegranskat)abstract
    • Novel approaches are required in the clinical management of peripheral nerve injuries because current surgical techniques result in deficient sensory recovery. Microsurgery alone fails to address extensive cell death in the dorsal root ganglia (DRG), in addition to poor axonal regeneration. Incorporation of cultured cells into nerve conduits may offer a novel approach in which to combine nerve repair and enhance axonal regeneration with neuroprotective therapies. We examined apoptotic mediator expression in rat DRG neurons following repair of a 10-mm sciatic nerve gap using a novel synthetic conduit made of poly epsilon-caprolactone (PCL) and primed with adipose-derived stem cells (ADSC) differentiated towards a Schwann cell phenotype or with primary adult Schwann cells. Differentiated ADSC expressed a range of neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and neurotrophin-4 (NT4). Incorporation of either differentiated ADSC or Schwann cells significantly increased anti-apoptotic Bcl-2 mRNA expression (P<0.001) in the DRG, while significantly decreasing proapoptotic Bax (P<0.001) and caspase-3 mRNA (P<0.01) expression. Cleaved caspase-3 protein was observed in the DRG following nerve injury which was attenuated when nerve repair was performed using conduits seeded with cells. Cell incorporation into conduit repair of peripheral nerves demonstrates experimental promise as a novel intervention to prevent DRG neuronal loss. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  •  
37.
  • Reid, Adam J, et al. (författare)
  • Peripherin and ATF3 genes are differentially regulated in regenerating and non-regenerating primary sensory neurons
  • 2010
  • Ingår i: Brain Research. - : Elsevier. - 0006-8993 .- 1872-6240. ; 1310, s. 1-7
  • Tidskriftsartikel (refereegranskat)abstract
    • Peripheral nerve injury leads to deficient recovery of sensation and a causative factor may be that only 50-60% of primary sensory neurons succeed in regenerating axons after primary nerve repair. In this study, an in vivo rat sciatic nerve injury and regeneration model was combined with laser microdissection and quantitative real-time polymerase chain reaction with the aim of examining the gene expression of regenerative molecules in cutaneous and muscular sensory neurons. Recent studies have identified peripherin and ATF-3 molecules as crucial for neurite outgrowth propagation; our novel findings demonstrate a subpopulation of non-regenerating sensory neurons characterized by a failure to upregulate transcription of these molecules and that a greater peripherin mRNA expression in injured cutaneous neurons may potentiate this subpopulation to regenerate more axons than muscle afferent neurons following injury. The gene expression of the structural neurofilament NF-H is found to be significantly downregulated following injury in both sensory subpopulations.
  •  
38.
  • Reid, Adam J, et al. (författare)
  • Phenotype of distinct primary sensory afferent subpopulations and caspase-3 expression following axotomy
  • 2011
  • Ingår i: Histochemistry and Cell Biology. - : Springer Science and Business Media LLC. - 0948-6143 .- 1432-119X. ; 136:1, s. 71-78
  • Tidskriftsartikel (refereegranskat)abstract
    • Specific sensory neuronal subpopulations show contrasting responses to peripheral nerve injury, as shown by the axotomy-induced death of many cutaneous sensory neurons whilst muscular sensory afferents survive an identical insult. We used a novel combination of retrograde neuronal tracing with immunohistochemistry and laser microdissection techniques, in order to describe the neurochemistry of medial gastrocnemius (muscular sensory afferents) and sural (cutaneous sensory afferents) branches of the rat sciatic nerve and relate this to the pro-apoptotic caspase-3 gene expression following nerve transection. Our results demonstrated distinctions in medial gastrocnemius and sural neuron populations with the most striking difference in the respective proportions of isolectin B4 (IB4) staining neurons (3.7 V 32.8%). The mean neuronal area of the medial gastrocnemius (MG) neurons was larger than that of the sural (SUR) neurons (1,070.8 V 646.2 μm²) and each phenotypic group was significantly smaller in sural neurons than in MG neurons. At 1 week post-axotomy, MG neurons markedly downregulated caspase-3, whilst SUR neurons upregulated caspase-3 gene expression; this may be attributable to the differing IB4-positive composition of the subpopulations. These findings provide further clarification in the understanding of two distinct neuronal populations used increasingly in nerve injury models.
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39.
  • Sun, M, et al. (författare)
  • In vitro and in vivo testing of novel ultrathin PCL and PCL/PLA blend films as peripheral nerve conduit
  • 2010
  • Ingår i: Journal of Biomedical Materials Research. Part A. - : Wiley. - 1549-3296 .- 1552-4965. ; 93:4, s. 1470-1481
  • Tidskriftsartikel (refereegranskat)abstract
    • In an attempt to obviate the drawbacks of nerve autograft, ultrathin microporous biodegradable PCL and PCL/PLA films were tested for their compatibility with motor neuron-like NG108-15 cells and primary Schwann cells. Data obtained from MTS colorimetric and DNA fluorimetric assays showed that both cell lines readily attached and proliferated on these materials. Images taken using scanning electron microscope and fluorescence microscope confirmed these observations. Enhanced cell-surface interaction was achieved by pretreating the films in NaOH solution. Importantly, NG108-15 cells could be induced into differentiated phenotype with long, un-branched neurites growing across the surface of the materials. The bipolar spindle-shaped phenotype of Schwann cells was also retained on these scaffolds. Positive immunochemical staining using antibodies against neurofilament for NG108-15 cells and S100 for Schwann cells indicated the expression of these marker proteins. In a small-scaled pilot testing, the performance of PCL conduits in bridging up a 10 mm gap in rat sciatic nerve model was assessed. Immunohistochemical staining showed that regenerated nerve tissue and penetrated Schwann cells have the potential to span the whole length of the conduit in 2 weeks.
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40.
  • Sun, Mingzhu, et al. (författare)
  • Novel thin-walled nerve conduit with microgrooved surface patterns for enhanced peripheral nerve repair
  • 2010
  • Ingår i: Journal of materials science. Materials in medicine. - : Springer. - 0957-4530 .- 1573-4838. ; 21:10, s. 2765-2774
  • Tidskriftsartikel (refereegranskat)abstract
    • Randomly aligned nerve cells in vitro on conventional culture substrata do not represent the complex neuronal network in vivo and neurites growing in uncontrolled manner may form neuroma. It is of great importance to mimic the organised growth pattern of nerve cells in the study of peripheral nerve repair. The aim of this work was to modify and optimize the photolithographic technique in creating a reusable template in the form of a silicon wafer that could be used to produce contact guidance on biodegradable polymer surface for the orientated growth of nerve cells. Micro-grooves (approximately 3 μm in depth) were etched into the silicon template using KOH at increased temperature. The originality of this work lies in the low cost and high efficiency method in producing microgrooves on the surface of biodegradable ultra-thin polymer substrates (50-100 μm), which can be readily rolled up to form clinically implantable nerve conduits. The design of a pattern with small ridge width (i.e., 5 μm) and bigger groove width (i.e., 20 μm) favored the alignment of cells along the grooves rather than on the ridges of the patterns, which minimized the effect of cross growing of neurites between adjacent grooves. Effectively, enhanced nerve regeneration could be anticipated from these patterned conduits.
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41.
  • Terenghi, Giorgio, et al. (författare)
  • Chapter 21 : Use of stem cells for improving nerve regeneration
  • 2009
  • Ingår i: International review of neurobiology. - 0074-7742 .- 2162-5514. ; 87, s. 393-403
  • Forskningsöversikt (refereegranskat)abstract
    • A clear need exists for new surgical approaches to enhance the recuperation of functions after peripheral nerve injury and repair. At present, advances in the regenerative medicine fields of biomaterials, cellular engineering, and molecular biology are all contributing to the development of a bioengineered nerve implant, which could be used clinically as an alternative to nerve autograft. In this review we examine the recent progress in this field, looking in particular at the applicability of Schwann cells and stem cell transplantation to enhance nerve regeneration.
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42.
  • Terenghi, Giorgio, et al. (författare)
  • The nerve injury and the dying neurons : diagnosis and prevention
  • 2011
  • Ingår i: Journal of hand surgery. European volume. - Oxford : Churchill Livingstone. - 1753-1934 .- 2043-6289. ; 36E:9, s. 730-734
  • Forskningsöversikt (refereegranskat)abstract
    • Following distal nerve injury significant sensory neuronal cell death occurs in the dorsal root ganglia, while after a more proximal injury, such as brachial plexus injury, a sizeable proportion of spinal motoneurons also undergo cell death. This phenomenon has been undervalued for a long time, but it has a significant role in the lack of functional recuperation, as neuronal cells cannot divide and be replaced, hence the resulting nerve regeneration is usually suboptimal. It is now accepted that this cell death is due to apoptosis, as indicated by analysis of specific genes involved in the apoptotic signalling cascade. Immediate nerve repair, either by direct suturing or nerve grafting, gives a degree of neuroprotection, but this approach does not fully prevent neuronal cell death and importantly it is not always possible. Our work has shown that pharmacological intervention using either acetyl-L-carnitine (ALCAR) or N-acetyl-cysteine (NAC) give complete neuroprotection in different types of peripheral nerve injury. Both compounds are clinically safe and experimental work has defined the best dose, timing after injury and duration of administration. The efficacy of neuroprotection of ALCAR and NAC can be monitored non-invasively using MRI, as demonstrated experimentally and more recently by clinical studies of the volume of dorsal root ganglia. Translation to patients of this pharmacological intervention requires further work, but the available results indicate that this approach will help to secure a better functional outcome following peripheral nerve injury and repair.
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43.
  • Thornton, Martin R, et al. (författare)
  • Neurotrophins 3 and 4 differentially regulate NCAM, L1 and N-cadherin expression during peripheral nerve regeneration.
  • 2008
  • Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 49:Pt 2, s. 165-74
  • Tidskriftsartikel (refereegranskat)abstract
    • The addition of NT-3 (neurotrophin 3) or NT-4 to injured nerves improves their regeneration potential and may aid axon guidance. It is not well defined whether NTs (neurotrophins) influence other elements, such as the cell-adhesion molecules, which promote nerve guidance and regeneration. Using poly-3-hydroxybutyrate conduits, we applied either NT-3 or NT-4 to axotomized rat sciatic nerves and monitored nerve regeneration and cell-adhesion molecule expression. Regenerating nerves were stained with antibodies against NCAM (neural cell-adhesion molecule) and N-cadherin 2 weeks after injury and staining intensity was quantified. NCAM, N-cadherin and L1 (L1 cell-adhesion molecule) transcription was measured in the proximal and distal stumps and ipsilateral DRG (dorsal root ganglia) (fourth and fifth DRG) using RT (reverse transcriptase)-PCR. Both NT-3 and NT-4 increased NCAM and L1 transcript levels in the DRG of axotomized nerves. This is reflected in the increased NCAM expression at the proximal stump and regeneration front. Increased levels of NCAM were also observed in the distal stump. NT-4 administration increased N-cadherin levels proximal to the injury, but not distally. Following NT-3 administration, N-cadherin expression decreased in proximal and distal stumps compared with the control. In conclusion, NTs differentially alter adhesion molecule expression in regenerating nerves and transcription in the corresponding DRG, although these changes in expression do not alter NT-enhanced regeneration. Thus we propose that retrograde transport of the NTs to the DRG affects adhesion molecule transcription, reflected by protein expression in peripheral nerve axons.
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44.
  • Tohill, Mel P, et al. (författare)
  • Green fluorescent protein is a stable morphological marker for schwann cell transplants in bioengineered nerve conduits
  • 2004
  • Ingår i: Tissue engineering. - Larchmont, NY : Mary Ann Liebert, Inc.. - 1076-3279 .- 1557-8690. ; 10:9-10, s. 1359-1367
  • Tidskriftsartikel (refereegranskat)abstract
    • Bioengineered systems incorporate cultured cells to mimic the substituted tissue. A labeling method is necessary to monitor the survival of transplanted cells within the host. This labeling method must be compatible with the histochemical methods used for morphological analysis. This study assessed (1) The in vitro characteristics of Schwann cells (SCs) labeled with green fluorescent protein (GFP), (2) the in vivo effect of transplanted GFP-SCs in a model of peripheral nerve injury, and (3) the compatibility of GFP-SCs with immunofluorescence histochemical techniques. SCs were retrovirally labeled with GFP and their growth characteristics were compared with those of nontransduced SCs (ntSCs). GFP-SCs were seeded in a resorbable nerve conduit for grafting into a 1-cm gap in rat sciatic nerve. Grafts were harvested after 2 weeks and immunofluorescent staining was performed to measure axonal and SC regeneration distances and to identify GFP-SCs. Results of GFP-SC vitality assays did not vary significantly from those of ntSC assays. GFP-SCs were readily located ex vivo and stimulated significantly better axonal and SC regeneration distances in comparison with empty conduits. These findings show that GFP labeling does not have a deleterious effect on SCs and that it is a useful labeling method for the study of bioengineered systems.
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45.
  • Tohill, Mel, et al. (författare)
  • Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration
  • 2004
  • Ingår i: Neuroscience Letters. - : Elsevier. - 0304-3940 .- 1872-7972. ; 362:3, s. 200-203
  • Tidskriftsartikel (refereegranskat)abstract
    • Bone marrow mesenchymal stem cells can trans-differentiate into neuronal phenotypes. We examined the differentiation of marrow stromal cells (MSCs) in culture and during nerve regeneration. MSCs from adult rats were exposed to glial growth factor (GGF) to stimulate glial differentiation. Subsequently differentiated MSCs were retrovirally labelled with green fluorescent protein and transplanted into 1 cm nerve conduits in the rat sciatic nerve. Fifteen days post-operatively the conduits were examined for axonal and Schwann cell regeneration and MSC integration. In vitro, MSCs exposed to GGF expressed S100 and glial fibrillary acidic protein. Following transplantation, MSCs maintained S100 expression and enhanced nerve regeneration, with significant Schwann cell regeneration compared to control (2.7 +/- 0.21 vs. 2.05 +/- .21 mm; P < 0.05). MSCs not exposed to GGF prior to transplantation expressed S100 in vivo indicating glial differentiation in response to local cytokines and growth factors.
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46.
  • Tse, Kai-Hei, et al. (författare)
  • In vitro evaluation of polyester-based scaffolds seeded with adipose derived stem cells for peripheral nerve regeneration
  • 2010
  • Ingår i: Journal of Biomedical Materials Research. Part A. - : Wiley. - 1549-3296 .- 1552-4965. ; 95:3, s. 701-708
  • Tidskriftsartikel (refereegranskat)abstract
    • To overcome the disadvantages of autografts for peripheral nerve repair, different methods such as artificial nerve conduits have been investigated for an alternative approach. This study demonstrated that solvent casting is a simple but efficient method to create thin polyester-based scaffolds for stem cell delivery. Using poly (ε-caprolactone) and poly (D,L-lactic acid), we produced scaffold films containing heterogenous depressions (pits) on the air surface with a size ranging from 0.5 to 30 μm(2). These scaffolds were moderately hydrophobic; however, they supported the differentiation of adipose derived stem cells (ADSC) into a Schwann cell-like phenotype. The differentiated ADSC (dADSC) expressed S100 protein and glial fibrillary acidic protein and readily adhered to the films and proliferated at a similar rate to those cultured on tissue culture polystyrene. Cells were also positive for proliferating cell nuclear antigen. Furthermore, dADSC retained functional activity and significantly enhanced neurite outgrowth from dorsal root ganglia neurons. This study suggests polymer scaffolds combined with dADSCs could be a promising therapy for peripheral nerve injuries.
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47.
  • West, Christian A, et al. (författare)
  • Analysis of the dose-response of N-acetylcysteine in the prevention of sensory neuronal loss after peripheral nerve injury
  • 2007
  • Ingår i: How to Improve the Results of Peripheral Nerve Surgery. - Vienna : Springer. - 9783211729557 - 9783211729588 ; , s. 29-31
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • BACKGROUND: N-Acetylcysteine (NAC) is a safe pharmaceutical agent known to protect cells from oxidative damage. Following peripheral nerve transection, NAC has been found to eliminate sensory neuronal loss. This study examines the dose-response relationship of NAC in preventing neuronal death. METHODS AND FINDINGS: The rat sciatic nerve transection model was used, and stereological quantification of sensory neuron survival carried out at two weeks post-axotomy. NAC was administered systemically as an intraperitoneal injection to five groups of rats at a range of doses (1-300 mg/kg/day). Significant neuronal loss was observed in the 1 mg/kg/day dosage group (18.5% loss, p = 0.067 vs. sham treatment). A degree of neuroprotection occurred with 10 mg/kg/day (9.1% loss, p < 0.005 vs. control), whilst there was no significant loss with either 150 or 300 mg/kg/day. CONCLUSIONS: The prevention of sensory neuronal loss with NAC is dose dependent and effective over a wide therapeutic range. This analysis confirms the efficacy of systemic administration and provides a dose framework with which NAC has clinical potential to improve outcome after peripheral nerve trauma.
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48.
  • West, Christian Alexander, et al. (författare)
  • Sensory Neurons of the Human Brachial Plexus : A Quantitative Study Employing Optical Fractionation and In-Vivo Volumetric Magnetic Resonance Imaging.
  • 2012
  • Ingår i: Neurosurgery. - Philadelphia : Lippincott Williams & Wilkins. - 0148-396X .- 1524-4040. ; 70:5, s. 1183-1194
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: Extensive neuron death following peripheral nerve trauma is implicated in poor sensory recovery. Translational research for experimentally proven neuroprotective drugs requires knowledge of the numbers and distribution of sensory neurons in the human upper limb, and a novel non-invasive clinical measure of neuron loss. OBJECTIVE: To compare optical fractionation and volumetric MRI of dorsal root ganglia (DRG) in histological quantification and objective clinical assessment of human brachial plexus sensory neurons. METHODS: Bilateral C5-T1 DRG were harvested from 5 human cadavers for stereological volume measurement and sensory neuron counts (optical fractionator). MRI scans were obtained from 14 normal volunteers for volumetric analysis of C5-T1 DRG. RESULTS: 425,409 (SD 15,596) sensory neurons innervate the brachial plexus with a significant difference in neuron counts and DRG volume between segmental levels (p<0.0001), with C7 ganglion containing the most. DRG volume correlated with neuron counts (r=0.75, p<.001). Vertebral artery pulsation hindered C5&6 imaging, yet high resolution MRI of C7, C8 and T1 DRG permitted unbiased volume measurement. In accord with histological analysis, MRI confirmed a significant difference between C7, C8 and T1 DRG volume (p<.001), inter-individual variability (COV 15.3%), and sex differences (p=.04). Slight right-left (R/L) sided disparity in neuron counts (2.5%, p=.04) was possibly related to hand dominance, but no significant volume disparity existed. CONCLUSION: Neuron counts for the human brachial plexus are presented. These correlate with histological DRG volumes and concur with volumetric MRI results in human volunteers. Volumetric MRI of C7-T1 DRG is a legitimate non-invasive proxy measure of sensory neurons for clinical study.
  •  
49.
  • West, Christian A, et al. (författare)
  • Volumetric magnetic resonance imaging of dorsal root ganglia for the objective quantitative assessment of neuron death after peripheral nerve injury.
  • 2007
  • Ingår i: Experimental Neurology. - : Elsevier BV. - 0014-4886 .- 1090-2430. ; 203:1, s. 22-33
  • Tidskriftsartikel (refereegranskat)abstract
    • Prevention of neuron death after peripheral nerve injury is vital to regaining adequate cutaneous innervation density and quality of sensation, and while experimentally proven neuroprotective therapies exist, there lacks suitable clinical outcome measures for translational research. Axotomized dorsal root ganglia (DRG) histologically exhibit volume reduction in proportion to the amount of neuronal death within them. Hence, this study evaluated the validity of using magnetic resonance imaging (MRI) to quantify DRG volume as a proxy measure of cell death. A high-resolution 3D MRI sequence was developed for volumetric quantification of the L4 DRG in the rat sciatic nerve model. An unoperated "control" group (n=4), and a "nerve transection" group (n=6), 4 weeks after axotomy, were scanned. Accuracy and validity of the technique were evaluated by comparison with morphological quantification of DRG volume and stereological counts of surviving neurons (optical fractionator). The technique was precise (coefficient of variation=4.3%), highly repeatable (9% variability), and sensitive (mean 15.0% volume reduction in axotomized ganglia detected with statistical significance: p<0.01). MRI showed strong and highly significant correlation with morphological measures of DRG volume loss (r=0.90, p<0.001), which in turn correlated well with neuron loss (r=0.75, p<0.05). MRI similarly exhibited direct correlation with neuron loss (r=0.67, p<0.05) with consistent agreement. MRI volumetric quantification of DRG is therefore a valid in vivo measure of neuron loss. As a non-invasive, objective measure of neuronal death after nerve trauma this technique has potential as a diagnostic modality and a quantitative tool for clinical studies of neuroprotective agents.
  •  
50.
  • Wilson, Andrew D H, et al. (författare)
  • Acetyl-l-carnitine increases nerve regeneration and target organ reinnervation : A morphological study
  • 2010
  • Ingår i: Journal of Plastic, Reconstructive & Aesthetic Surgery. - : Elsevier. - 1748-6815 .- 1878-0539. ; 63:7, s. 1186-1195
  • Tidskriftsartikel (refereegranskat)abstract
    • Peripheral nerve injury frequently results in functional morbidity since standard management fails to adequately address many of the neurobiological hurdles to optimal regeneration. Neuronal survival and regeneration are neurotrophin dependent and require increased aerobic capacity. Acetyl-l-carnitine (ALCAR) facilitates this need and prevents neuronal loss. ALCAR is clinically safe and is shown here to significantly improve nerve regeneration and target organ reinnervation. Two groups of five rats underwent sciatic nerve division followed by immediate repair. One group received parenteral ALCAR (50mg/kg/day) from time of operation until termination at 12 weeks. A 'sham treatment' group received normal saline. A third group was left unoperated and did not receive any treatment. A segment of nerve was harvested between 5mm proximal and 10mm distal to the repair in operated groups, and at the corresponding level in the unoperated group. Mean axonal count in normal, non-axotomised nerve was 14,720 (SD 2378). That of the saline group (17,217 SD 1808) was not significantly different from normal nerve (P=0.0985). Mean number of myelinated axons in the ALCAR group (24,460 SD 3750) was significantly greater than both sham group (P<0.01) and normal nerve (P=0.0012). Mean myelin thickness in the saline treated group (0.408mum SD 0.067mum) was less than normal nerve (0.770mum SD 0.143mum) (P<0.001). Mean myelin thickness in the ALCAR group (0.627mum SD 0.052mum) was greater than the sham (saline) group (P<0.01) and not statistically different from normal nerve (P=0.07). ALCAR increased dermal PGP9.5 staining by 210% compared to sham treatment (P<0.0001) and significantly reduced the mean percentage weight loss in gastrocnemius muscle (ALCAR group 0.203% vs. 0.312% in sham group P=0.015). ALCAR not only increases the number of regenerating nerve fibres but also morphologically improves the quality of regeneration and target organ reinnervation. Adjuvant ALCAR treatment may improve both sensory and motor outcomes and merits further investigation.
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