Giles W Plant

All Publications

• Sensory and descending motor circuitry during development and injury. Current opinion in neurobiology Plant, G. W., Weinrich, J. A., Kaltschmidt, J. A. 2018; 53: 156–61

  Abstract

  Proprioceptive sensory input and descending supraspinal projections are two major inputs that feed into and influence spinal circuitry and locomotor behaviors. Here we review their influence on each other during development and after spinal cord injury. We highlight developmental mechanisms of circuit formation as they relate to the sensory-motor circuit and its reciprocal interactions with local spinal interneurons, as well as competitive interactions between proprioceptive and descending supraspinal inputs in the setting of spinal cord injury.

  View details for DOI 10.1016/j.conb.2018.08.008

  View details for PubMedID 30205323

• Viral Transduction of Schwann Cells for Peripheral Nerve Repair. Methods in molecular biology (Clifton, N.J.) Plant, C. D., Plant, G. W. 2018; 1739: 455–66

  Abstract

  Schwann cells are the primary inducers of regeneration of the peripheral nervous system. Schwann cells can be isolated from adult peripheral nerves, expanded in large numbers, and genetically transduced by viral vectors in vitro prior to their use in vivo. Here we describe how to use lentiviral vectors to transduce primary Schwann cells in vitro. We also describe how cultured Schwann cells can be used in conjunction with decellularized peripheral nerve sheaths prepared by multiple freeze thawing of peripheral nerve tissue. This process depletes all native cells from the nerve sheath but maintains basal lamina integrity and flexibility. A major advantage of using these decellularized nerve sheaths in repair strategies is that they can be obtained from cadaveric tissue and therefore do not require patient matching because the immune response is generated from the intrinsic cells and not the sheath itself. The patient's own cells can then be used to repopulate the decellularized peripheral nerve sheath. Our technique described in this chapter uses decellularized nerve sheaths which are repopulated with extrinsic Schwann cells previously grown in vitro. The Schwann cells can also be engineered in multiple ways, for example, to secrete bioactive proteins beneficial to axonal regeneration.

  View details for DOI 10.1007/978-1-4939-7649-2_30

  View details for PubMedID 29546726

• Wnt/β-catenin signaling regulates ependymal cell development and adult homeostasis. Proceedings of the National Academy of Sciences of the United States of America Xing, L., Anbarchian, T., Tsai, J. M., Plant, G. W., Nusse, R. 2018

  Abstract

  In the adult mouse spinal cord, the ependymal cell population that surrounds the central canal is thought to be a promising source of quiescent stem cells to treat spinal cord injury. Relatively little is known about the cellular origin of ependymal cells during spinal cord development, or the molecular mechanisms that regulate ependymal cells during adult homeostasis. Using genetic lineage tracing based on the Wnt target gene Axin2, we have characterized Wnt-responsive cells during spinal cord development. Our results revealed that Wnt-responsive progenitor cells are restricted to the dorsal midline throughout spinal cord development, which gives rise to dorsal ependymal cells in a spatially restricted pattern. This is contrary to previous reports that suggested an exclusively ventral origin of ependymal cells, suggesting that ependymal cells may retain positional identities in relation to their neural progenitors. Our results further demonstrated that in the postnatal and adult spinal cord, all ependymal cells express the Wnt/β-catenin signaling target gene Axin2, as well as Wnt ligands. Genetic elimination of β-catenin or inhibition of Wnt secretion in Axin2-expressing ependymal cells in vivo both resulted in impaired proliferation, indicating that Wnt/β-catenin signaling promotes ependymal cell proliferation. These results demonstrate the continued importance of Wnt/β-catenin signaling for both ependymal cell formation and regulation. By uncovering the molecular signals underlying the formation and regulation of spinal cord ependymal cells, our findings thus enable further targeting and manipulation of this promising source of quiescent stem cells for therapeutic interventions.

  View details for DOI 10.1073/pnas.1803297115

  View details for PubMedID 29891676

• Schwann Cell Transplantation Methods Using Biomaterials. Methods in molecular biology (Clifton, N.J.) Plant, C. D., Plant, G. W. 2018; 1739: 439–53

  Abstract

  Biomaterials can be utilized to assist in the transplantation of Schwann cells to the central and peripheral nervous system. The biomaterials can be natural or man-made, and can have preformed shapes or injectable formats. Biomaterials can play multiple roles in cellular transplantation; for example, they can assist with cellular integration and protect Schwann cells from cell death initiated by the lack of a substrate, an occurrence known as "anoikis." In addition, biomaterials can be engineered to increase cell proliferation and differentiation by the addition of ligands bound to the substrate. Here, we describe the incorporation of Schwann cells to both man-made and natural matrices for in vitro and in vivo measures relevant to Schwann cell transplantation strategies.

  View details for DOI 10.1007/978-1-4939-7649-2_29

  View details for PubMedID 29546725

• Recombinant biomaterials for treatment of spinal cord injuries Dubbin, K., Marquardt, L., Plant, G., Heilshorn, S. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000429556703370

• Intravenous Transplantation of Mesenchymal Progenitors Distribute Solely to the Lungs and Improve Outcomes in Cervical Spinal Cord Injury STEM CELLS White, S. V., Czisch, C. E., Han, M. H., Plant, C. D., Harvey, A. R., Plant, G. W. 2016; 34 (7): 1812-1825

  Abstract

  Cellular transplantation strategies utilizing intraspinal injection of mesenchymal progenitor cells have been reported as beneficial for spinal cord injuries. However, intraspinal injection is not only technically challenging, but requires invasive surgical procedures for patients. Therefore, we investigated the feasibility and potential benefits of non-invasive intravenous injection of mesenchymal progenitor cells in two models of cervical spinal cord injury, unilateral C5 contusion and complete unilateral C5 hemisection. Mesenchymal progenitor cells were isolated from GFP-luciferase transgenic mice compact bone (1x10(6) cells) or vehicle (HBSS) were intravenously injected via the tail vein at D1, D3, D7, D10 or D14. Transplanted mesenchymal progenitor cells were tracked via bioluminescence imaging. Live in vivo imaging data showed that intravenously injected mesenchymal progenitor cells accumulate in the lungs, confirmed by post-mortem bioluminescence signal - irrespective of the time of injection or injury model. The results showed a rapid, positive modulation of the inflammatory response providing protection to the injured spinal cord tissue. Histological processing of the lungs showed GFP(+) cells evenly distributed around the alveoli. We propose that injected cells can act as cellular target decoys to an immune system primed by injury, thereby lessening the inflammatory response at the injury site. We also propose that intravenous injected mesenchymal progenitor cells modulate the immune system via the lungs through secreted immune mediators or contact interaction with peripheral organs. In conclusion, the timing of intravenous injection of mesenchymal progenitor cells is key to the success for improving function and tissue preservation following cervical spinal cord injury. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/stem.2364

  View details for Web of Science ID 000379902900009

  View details for PubMedID 26989838

• Induced Pluripotent Stem Cell Therapies for Cervical Spinal Cord Injury. International journal of molecular sciences Doulames, V. M., Plant, G. W. 2016; 17 (4)

  Abstract

  Cervical-level injuries account for the majority of presented spinal cord injuries (SCIs) to date. Despite the increase in survival rates due to emergency medicine improvements, overall quality of life remains poor, with patients facing variable deficits in respiratory and motor function. Therapies aiming to ameliorate symptoms and restore function, even partially, are urgently needed. Current therapeutic avenues in SCI seek to increase regenerative capacities through trophic and immunomodulatory factors, provide scaffolding to bridge the lesion site and promote regeneration of native axons, and to replace SCI-lost neurons and glia via intraspinal transplantation. Induced pluripotent stem cells (iPSCs) are a clinically viable means to accomplish this; they have no major ethical barriers, sources can be patient-matched and collected using non-invasive methods. In addition, the patient's own cells can be used to establish a starter population capable of producing multiple cell types. To date, there is only a limited pool of research examining iPSC-derived transplants in SCI-even less research that is specific to cervical injury. The purpose of the review herein is to explore both preclinical and clinical recent advances in iPSC therapies with a detailed focus on cervical spinal cord injury.

  View details for DOI 10.3390/ijms17040530

  View details for PubMedID 27070598

• Deficiency in matrix metalloproteinase-2 results in long-term vascular instability and regression in the injured mouse spinal cord. Experimental neurology Trivedi, A., Zhang, H., Ekeledo, A., Lee, S., Werb, Z., Plant, G. W., Noble-Haeusslein, L. J. 2016; 284 (Pt A): 50–62

  Abstract

  Angiogenesis plays a critical role in wound healing after spinal cord injury. Therefore, understanding the events that regulate angiogenesis has considerable relevance from a therapeutic standpoint. We evaluated the contribution of matrix metalloproteinase (MMP)-2 to angiogenesis and vascular stability in spinal cord injured MMP-2 knockout and wildtype (WT) littermates. While MMP-2 deficiency resulted in reduced endothelial cell division within the lesioned epicenter, there were no genotypic differences in vascularity (vascular density, vascular area, and endothelial cell number) over the first two weeks post-injury. However, by 21days post-injury MMP-2 deficiency resulted in a sharp decline in vascularity, indicative of vascular regression. Complementary in vitro studies of brain capillary endothelial cells confirmed MMP-2 dependent proliferation and tube formation. As deficiency in MMP-2 led to prolonged MMP-9 expression in the injured spinal cord, we examined both short-term and long-term exposure to MMP-9 in vitro. While MMP-9 supported endothelial tube formation and proliferation, prolonged exposure resulted in loss of tubes, findings consistent with vascular regression. Vascular instability is frequently associated with pericyte dissociation and precedes vascular regression. Quantification of PDGFrβ+ pericyte coverage of mature vessels within the glial scar (the reactive gliosis zone), a known source of MMP-9, revealed reduced coverage in MMP-2 deficient animals. These findings suggest that acting in the absence of MMP-2, MMP-9 transiently supports angiogenesis during the early phase of wound healing while its prolonged expression leads to vascular instability and regression. These findings should be considered while developing therapeutic interventions that block MMPs.

  View details for DOI 10.1016/j.expneurol.2016.07.018

  View details for PubMedID 27468657

• Neural Placode Tissue Derived From Myelomeningocele Repair Serves as a Viable Source of Oligodendrocyte Progenitor Cells. Neurosurgery Mitra, S. S., Feroze, A. H., Gholamin, S., Richard, C., Esparza, R., Zhang, M., Azad, T. D., Alrfaei, B., Kahn, S. A., Hutter, G., Guzman, R., Creasey, G. H., Plant, G. W., Weissman, I. L., Edwards, M. S., Cheshier, S. 2015; 77 (5): 794-802

  Abstract

  The presence, characteristics, and potential clinical relevance of neural progenitor populations within the neural placodes of myelomeningocele patients remain to be studied. Neural stem cells are known to reside adjacent to ependyma-lined surfaces along the central nervous system axis.Given such neuroanatomic correlation and regenerative capacity in fetal development, we assessed myelomeningocele-derived neural placode tissue as a potentially novel source of neural stem and progenitor cells.Nonfunctional neural placode tissue was harvested from infants during the surgical repair of myelomeningocele and subsequently further analyzed by in vitro studies, flow cytometry, and immunofluorescence. To assess lineage potential, neural placode-derived neurospheres were subjected to differential media conditions. Through assessment of platelet-derived growth factor receptor α (PDGFRα) and CD15 cell marker expression, Sox2+Olig2+ putative oligodendrocyte progenitor cells were successfully isolated.PDGFRαCD15 cell populations demonstrated the highest rate of self-renewal capacity and multipotency of cell progeny. Immunofluorescence of neural placode-derived neurospheres demonstrated preferential expression of the oligodendrocyte progenitor marker, CNPase, whereas differentiation to neurons and astrocytes was also noted, albeit to a limited degree.Neural placode tissue contains multipotent progenitors that are preferentially biased toward oligodendrocyte progenitor cell differentiation and presents a novel source of such cells for use in the treatment of a variety of pediatric and adult neurological disease, including spinal cord injury, multiple sclerosis, and metabolic leukoencephalopathies.

  View details for DOI 10.1227/NEU.0000000000000918

  View details for PubMedID 26225855

• Geometrical versus Random beta-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior PLOS ONE Sweet, L., Kang, Y., Czisch, C., Witek, L., Shi, Y., Smay, J., Plant, G. W., Yang, Y. 2015; 10 (10)

  Abstract

  Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP) scaffolds arranged in 3D printed-lattice (P-β-TCP) and randomly-porous, template-casted (N-β-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf), neutrophin-3 (nt-3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  View details for DOI 10.1371/journal.pone.0139820

  View details for Web of Science ID 000362510600085

  View details for PubMedID 26444999

• Large animal and primate models of spinal cord injury for the testing of novel therapies EXPERIMENTAL NEUROLOGY Kwon, B. K., Streijger, F., Hill, C. E., Anderson, A. J., Bacon, M., Beattie, M. S., Blesch, A., Bradbury, E. J., Brown, A., Bresnahan, J. C., Case, C. C., Colburn, R. W., David, S., Fawcett, J. W., Ferguson, A. R., Fischer, I., Floyd, C. L., Gensel, J. C., Houle, J. D., Jakeman, L. B., Jeffery, N. D., Jones, L. A., Kleitman, N., Kocsis, J., Lu, P., Magnuson, D. S., Marsala, M., Moore, S. W., Mothe, A. J., Oudega, M., Plant, G. W., Rabchevsky, A. S., Schwab, J. M., Silver, J., Steward, O., Xu, X., Guest, J. D., Tetzlaff, W. 2015; 269: 154-168

  Abstract

  Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.

  View details for DOI 10.1016/j.expneurol.2015.04.008

  View details for Web of Science ID 000355640600015

  View details for PubMedID 25902036

• Hierarchical Patterning of Multifunctional Conducting Polymer Nanoparticles as a Bionic Platform for Topographic Contact Guidance ACS NANO Ho, D., Zou, J., Chen, X., Munshi, A., Smith, N. M., Agarwal, V., Hodgetts, S. I., Plant, G. W., Bakker, A. J., Harvey, A. R., Luzinov, I., Iyer, K. S. 2015; 9 (2): 1767-1774

  Abstract

  The use of programmed electrical signals to influence biological events has been a widely accepted clinical methodology for neurostimulation. An optimal biocompatible platform for neural activation efficiently transfers electrical signals across the electrode-cell interface and also incorporates large-area neural guidance conduits. Inherently conducting polymers (ICPs) have emerged as frontrunners as soft biocompatible alternatives to traditionally used metal electrodes, which are highly invasive and elicit tissue damage over long-term implantation. However, fabrication techniques for the ICPs suffer a major bottleneck, which limits their usability and medical translation. Herein, we report that these limitations can be overcome using colloidal chemistry to fabricate multimodal conducting polymer nanoparticles. Furthermore, we demonstrate that these polymer nanoparticles can be precisely assembled into large-area linear conduits using surface chemistry. Finally, we validate that this platform can act as guidance conduits for neurostimulation, whereby the presence of electrical current induces remarkable dendritic axonal sprouting of cells.

  View details for DOI 10.1021/nn506607x

  View details for Web of Science ID 000349940500072

  View details for PubMedID 25623615

• Changes in expression of Class 3 Semaphorins and their receptors during development of the rat retina and superior colliculus BMC DEVELOPMENTAL BIOLOGY Sharma, A., LeVaillant, C. J., Plant, G. W., Harvey, A. R. 2014; 14

  Abstract

  Members of the Semaphorin 3 family (Sema3s) influence the development of the central nervous system, and some are implicated in regulating aspects of visual system development. However, we lack information about the timing of expression of the Sema3s with respect to different developmental epochs in the mammalian visual system. In this time-course study in the rat, we document for the first time changes in the expression of RNAs for the majority of Class 3 Semaphorins (Sema3s) and their receptor components during the development of the rat retina and superior colliculus (SC).During retinal development, transcript levels changed for all of the Sema3s examined, as well as Nrp2, Plxna2, Plxna3, and Plxna4a. In the SC there were also changes in transcript levels for all Sema3s tested, as well as Nrp1, Nrp2, Plxna1, Plxna2, Plxna3, and Plxna4a. These changes correlate with well-established epochs, and our data suggest that the Sema3s could influence retinal ganglion cell (RGC) apoptosis, patterning and connectivity in the maturing retina and SC, and perhaps guidance of RGC and cortical axons in the SC. Functionally we found that SEMA3A, SEMA3C, SEMA3E, and SEMA3F proteins collapsed purified postnatal day 1 RGC growth cones in vitro. Significantly this is a developmental stage when RGCs are growing into and within the SC and are exposed to Sema3 ligands.These new data describing the overall temporal regulation of Sema3 expression in the rat retina and SC provide a platform for further work characterising the functional impact of these proteins on the development and maturation of mammalian visual pathways.

  View details for DOI 10.1186/s12861-014-0034-9

  View details for Web of Science ID 000339840900001

  View details for PubMedID 25062604

• Systematic Review of Induced Pluripotent Stem Cell Technology as a Potential Clinical Therapy for Spinal Cord Injury CELL TRANSPLANTATION Kramer, A. S., Harvey, A. R., Plant, G. W., Hodgetts, S. I. 2013; 22 (4): 571-617

  Abstract

  Transplantation therapies aimed at repairing neurodegenerative and neuropathological conditions of the central nervous system (CNS) have utilised and tested a variety of cell candidates, each with its own unique set of advantages and disadvantages. The use and popularity of each cell type is guided by a number of factors including the nature of the experimental model, neuroprotection capacity, the ability to promote plasticity and guided axonal growth, and the cells' myelination capability. The promise of stem cells, with their reported ability to give rise to neuronal lineages to replace lost endogenous cells and myelin, integrate into host tissue, restore functional connectivity and provide trophic support to enhance and direct intrinsic regenerative ability, has been seen as a most encouraging step forward. The advent of the induced pluripotent stem cell (iPSC), which represents the ability to "re-program" somatic cells into a pluripotent state, hails the arrival of a new cell transplantation candidate for potential clinical application in therapies designed to promote repair and/or regeneration of the CNS. Since the initial development of iPSC technology, these cells have been extensively characterised in vitro and in a number of pathological conditions, and were originally reported to be equivalent to embryonic stem cells (ESCs). This review highlights emerging evidence that suggests iPSCs are not necessarily indistinguishable from ESCs, and may occupy a different "state" of pluripotency with differences in gene expression, methylation patterns and genomic aberrations which may reflect incomplete reprogramming and may therefore impact on the regenerative potential of these donor cells in therapies. It also highlights the limitations of current technologies used to generate these cells. Moreover we provide a systematic review of the state of play with regard to the use of iPSCs in the treatment of neurodegenerative and neuropathological conditions. The importance of balancing the promise of this transplantation candidate in the light of these emerging properties is crucial as the potential application in the clinical setting approaches.The first of three sections in this review, discusses (A) the pathophysiology of spinal cord injury (SCI) and how stem cell therapies can positively alter the pathology in experimental SCI. Part B summarises (i) the available technologies to deliver transgenes to generate iPSCs and (ii) recent data comparing iPSCs to ESCs in terms of characteristics and molecular composition. Lastly, in (C) we evaluate iPSC-based therapies as a candidate to treat SCI on the basis of their neurite induction capability compared to embryonic stem cells, and provide a summary of available in vivo data of iPSCs used in SCI and other disease models.

  View details for DOI 10.3727/096368912X655208

  View details for Web of Science ID 000319639700002

  View details for PubMedID 22944020

• Immunohistochemical, Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF, CNTF or NT3. PloS one Godinho, M. J., Teh, L., Pollett, M. A., Goodman, D., Hodgetts, S. I., Sweetman, I., Walters, M., Verhaagen, J., Plant, G. W., Harvey, A. R. 2013; 8 (8)

  Abstract

  We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB4 or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.

  View details for DOI 10.1371/journal.pone.0069987

  View details for PubMedID 23950907

• Human Mesenchymal Precursor Cells (Stro-1(+)) From Spinal Cord Injury Patients Improve Functional Recovery and Tissue Sparing in an Acute Spinal Cord Injury Rat Model CELL TRANSPLANTATION Hodgetts, S. I., Simmons, P. J., Plant, G. W. 2013; 22 (3): 393-412

  Abstract

  This study aimed to determine the potential of purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) to repair the injured spinal cord (SC) after transplantation into T-cell-deficient athymic RNU nude rats following acute moderate contusive spinal cord injury (SCI). hMPCs were isolated from the bone marrow (BM) stroma of SCI patients and transplanted as a suspension graft in medium [with or without immunosuppression using cyclosporin A (CsA)]. Extensive anatomical analysis shows statistically significant improvement in functional recovery, tissue sparing, and cyst reduction. We provide quantitative assessment of supraspinal projections in combination with functional outcomes. hMPC-transplanted animals consistently achieved mean BBB scores of 15 at 8 weeks post injury. Quantitative histological staining revealed that graft-recipient animals possessed more intact spinal tissue and reduced cyst formation than controls. Fluorogold (FG) retrograde tracing revealed sparing/regeneration of supraspinal and local propriospinal axonal pathways, but no statistical differences were observed compared to controls. Immunohistochemical analysis revealed increased serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted donor hMPCs 2 weeks posttransplantation, but no evidence of hMPC transdifferentiation was seen. Although hMPCs initially survive at 2 weeks posttransplantation, their numbers were dramatically reduced and no cells were detected at 8 weeks posttransplantation using retroviral/lentiviral GFP labeling and a human nuclear antigen (HNA) antibody. Additional immunosuppression with CsA did not improve hMPC survival or their ability to promote tissue sparing or functional recovery. We propose Stro-1(+)-selected hMPCs provide (i) a reproducible source for stem cell transplantation for SC therapy and (ii) a positive host microenvironment resulting in the promotion of tissue sparing/repair that subsequently improves behavioral outcomes after SCI. Our results provide a new candidate for consideration as a stem cell therapy for the repair of traumatic CNS injury.

  View details for DOI 10.3727/096368912X656081

  View details for Web of Science ID 000317257000002

  View details for PubMedID 23007022

• Magnetic field directed fabrication of conducting polymer nanowires. Chemical communications (Cambridge, England) Ho, D., Peerzade, S. A., Becker, T., Hodgetts, S. I., Harvey, A. R., Plant, G. W., Woodward, R. C., Luzinov, I., St Pierre, T. G., Iyer, K. S. 2013; 49 (64): 7138–40

  Abstract

  The self-assembly of nanoparticles is an efficient and precise method to fabricate nanoscale devices. By manipulating iron oxide nanoparticles in suspension with an external field to form magnetically directed linear assemblies, we demonstrate the feasibility of using this structure to template the synthesis of PEDOT:PSS conducting polymer nanowires in suspension. Furthermore these conducting wires can be assembled on interdigitated electrodes to form an array of conducting nanowires.

  View details for DOI 10.1039/c3cc41523f

  View details for PubMedID 23831765

• A comparison of the behavioral and anatomical outcomes in sub-acute and chronic spinal cord injury models following treatment with human mesenchymal precursor cell transplantation and recombinant decorin. Experimental neurology Hodgetts, S. I., Simmons, P. J., Plant, G. W. 2013

  Abstract

  This study assessed the potential of highly purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) in combination with the anti-scarring protein decorin to repair the injured spinal cord (SC). Donor hMPCs isolated from spinal cord injury (SCI) patients were transplanted into athymic rats as a suspension graft, alone or after previous treatment with, core (decorin(core)) and proteoglycan (decorin(pro)) isoforms of purified human recombinant decorin. Decorin was delivered via mini-osmotic pumps for 14days following sub-acute (7day) or chronic (1month) SCI. hMPCs were delivered to the spinal cord at 3weeks or 6weeks after the initial injury at T9 level. Behavioral and anatomical analysis in this study showed statistically significant improvement in functional recovery, tissue sparing and cyst volume reduction following hMPC therapy. The combination of decorin infusion followed by hMPC therapy did not improve these measured outcomes over the use of cell therapy alone, in either sub-acute or chronic SCI regimes. However, decorin infusion did improve tissue sparing, reduce spinal tissue cavitation and increase transplanted cell survivability as compared to controls. Immunohistochemical analysis of spinal cord sections revealed differences in glial, neuronal and extracellular matrix molecule expression within each experimental group. hMPC transplanted spinal cords showed the increased presence of serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted hMPCs for up to 2months; however, no evidence of hMPC transdifferentiation into neuronal or glial phenotypes. The number of hMPCs was dramatically reduced overall, and no transplanted cells were detected at 8weeks post-injection using lentiviral GFP labeling and human nuclear antigen antibody labeling. The presence of recombinant decorin in the cell transplantation regimes delayed in part the loss of donor cells, with small numbers remaining at 2months after transplantation. In vitro co-culture experiments with embryonic dorsal root ganglion explants revealed the growth promoting properties of hMPCs. Decorin did not increase axonal outgrowth from that achieved by hMPCs. We provide evidence for the first time that (Stro-1(+)) hMPCs provide: i) an advantageous source of allografts for stem cell transplantation for sub-acute and chronic spinal cord therapy, and (ii) a positive host microenvironment that promotes tissue sparing/repair that subsequently improves behavioral outcomes after SCI. This was not measurably improved by recombinant decorin treatment, but does provide important information for the future development and potential use of decorin in contusive SCI therapy.

  View details for DOI 10.1016/j.expneurol.2013.06.018

  View details for PubMedID 23867131

• Changes in mRNA Expression of Class 3 Semaphorins and Their Receptors in the Adult Rat Retino-Collicular System after Unilateral Optic Nerve Injury INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Sharma, A., Pollett, M. A., Plant, G. W., Harvey, A. R. 2012; 53 (13): 8367-8377

  Abstract

  Increasing interest in the role of Class 3 Semaphorins (Sema3s) in plasticity and repair in the injured mammalian central nervous system prompted us to characterize changes in Sema3 expression after optic nerve (ON) injury.We used unilateral ON transection (ONT) and ON crush (ONC) models in conjunction with quantitative polymerase chain reaction (qPCR), and in situ hybridization (ISH) to characterize postinjury changes in the expression of the Sema3s and their receptors in the rat retina, optic nerve, and superior colliculus (SC).We observed no changes in mRNA expression in axotomized retinas at 1 or 14 days after ONT, but there was a transient increase for Sema3b, Sema3f, L1cam, and Plxna3 at 3 days postinjury. There was no change in transcript expression in the deafferented contralateral SC 1 day following ONT, but there was a transient increase in Plxna2 at 3 days, and a decrease in Sema3e, L1cam, and Plxna4a mRNA levels by 14 days. There were also several changes in transcript expression in the unlesioned contralateral retina and ipsilateral SC that differed from those seen in axotomized retina and contralateral SC. At the injury site after ONC, there was a reduction in Sema3b and Sema3f mRNA at 6 hours, returning to control levels by 1 day, and a transient increase in SEMA3A immunoreactivity at 6 hours.These new data on Class 3 Semaphorins and their receptors provide more information about the complex reactive events that occur bilaterally in the retino-collicular system following unilateral adult ON injury.

  View details for DOI 10.1167/iovs.12-10799

  View details for Web of Science ID 000313056000060

  View details for PubMedID 23139269

• Scaffolds to promote spinal cord regeneration. Handbook of clinical neurology Sakiyama-Elbert, S., Johnson, P. J., Hodgetts, S. I., Plant, G. W., Harvey, A. R. 2012; 109: 575-594

  Abstract

  Substantial research effort in the spinal cord injury (SCI) field is directed towards reduction of secondary injury changes and enhancement of tissue sparing. However, pathway repair after complete transections, large lesions, or after chronic injury may require the implantation of some form of oriented bridging structure to restore tissue continuity across a trauma zone. These matrices or scaffolds should be biocompatible and create an environment that facilitates tissue growth and vascularization, and allow axons to regenerate through and beyond the implant in order to reconnect with "normal" tissue distal to the injury. The myelination of regrown axons is another important requirement. In this chapter, we describe recent advances in biomaterial technology designed to provide a terrain for regenerating axons to grow across the site of injury and/or create an environment for endogenous repair. Many different types of scaffold are under investigation; they can be biodegradable or nondegradable, natural or synthetic. Scaffolds can be designed to incorporate immobilized signaling molecules and/or used as devices for controlled release of therapeutic agents, including growth factors. These bridging structures can also be infiltrated with specific cell types deemed suitable for spinal cord repair.

  View details for DOI 10.1016/B978-0-444-52137-8.00036-X

  View details for PubMedID 23098738

• Embryonic-derived olfactory ensheathing cells remyelinate focal areas of spinal cord demyelination more efficiently than neonatal or adult derived cells. Cell transplantation Coutts, D. J., Humphries, C. E., Zhao, C., Plant, G. W., Franklin, R. J. 2012

  Abstract

  Transplanted olfactory ensheathing cells (OECs) contribute to functional recovery in a range of CNS injuries by several mechanisms, one of which is potentially their ability to form myelin sheaths. OECs sourced from donors of different ages have been shown to remyelinate in several in vitro and in vivo models. However, the optimal donor age for OEC associated remyelination is unclear. This project directly compared the remyelinating potential of p75 purified OEC transplants from three donor ages. OECs were sourced from the olfactory bulbs of embryonic, neonatal and adult rats, purified by immunopanning and their remyelinating potential directly compared by transplantation into the same adult rat toxin-induced model of spinal cord demyelination. Remyelination efficiency three weeks after transplant was assessed morphologically and by immunostaining. Our results indicate that all donor ages remyelinate, however this process is most efficiently achieved by embryonic-derived OECs.

  View details for DOI 10.3727/096368912X656153

  View details for PubMedID 23031825

• Olfactory ensheathing glia: Repairing injury to the mammalian visual system EXPERIMENTAL NEUROLOGY Plant, G. W., Harvey, A. R., Leaver, S. G., Lee, S. V. 2011; 229 (1): 99-108

  Abstract

  The visual system is widely used as a model in which to study neurotrauma of the central nervous system and to assess the effects of experimental therapies. Adult mammalian retinal ganglion cell axons do not normally regenerate their axons for long distances following injury. Trauma to the visual system, particularly damage to the optic nerve or central visual tracts, causes loss of electrical communication between the retina and visual processing areas in the brain. After optic nerve crush or transection, axons degenerate and retinal ganglion cells (RGCs) are lost over a period of days. To promote and maintain axonal growth and connectivity, strategies must be developed to limit RGC death and provide regenerating axons with permissive substrates and a sustainable growth milieu that will ultimately provide long term visual function. This review explores the role olfactory glia can play in this repair. We describe the isolation of these cells from the olfactory system, transplantation to the brain, gene therapy and the possible benefits that these cells may have over other cellular therapies to initiate repair, in particular the stimulation of axonal regeneration in visual pathways. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

  View details for DOI 10.1016/j.expneurol.2010.09.010

  View details for Web of Science ID 000290920300010

  View details for PubMedID 20850434

• Lack of fibulin-3 alters regenerative tissue responses in the primary olfactory pathway MATRIX BIOLOGY Vukovic, J., Marmorstein, L. Y., McLaughlin, P. J., Sasaki, T., Plant, G. W., Harvey, A. R., Ruitenberg, M. J. 2009; 28 (7): 406-415

  Abstract

  The adult olfactory epithelium has maintained the ability to reconstitute its olfactory sensory neurons (OSNs) from a basal progenitor cell compartment. This allows for life-long turnover and replacement of receptor components as well as repair of the primary olfactory pathway in response to injury and environmental insults. The present study investigated whether fibulin-3, a glycoprotein in the extracellular matrix and binding partner of tissue inhibitor of metalloproteinases-3 (TIMP-3), plays a role in ongoing plasticity and regenerative events in the adult primary olfactory pathway. In wild-type control mice, fibulin-3 protein was detected on IB4(+)CD31(+) blood vessels, nerve fascicles and the basement membrane underneath the olfactory epithelium. After target ablation (olfactory bulbectomy), fibulin-3 was also abundantly present in the central nervous system (CNS) scar tissue that occupied the bulbar cavity. Using two different lesion models, i.e. intranasal Triton X-100 lesion and olfactory bulbectomy, we show that fibulin-3 deficient (Efemp1(-/-)) mice have impaired recovery of the olfactory epithelium after injury. Ten days post-injury, Efemp1(-/-) mice showed altered basal stem/progenitor cell proliferation and increased overall numbers of mature (olfactory marker protein (OMP) -positive) versus immature OSNs. However, compromised regenerative capacity of the primary olfactory pathway in Efemp1(-/-) mice was evidenced by reduced numbers of mature OSNs at the later time point of 42 days post-injury. In addition to these neural differences there were consistent changes in blood vessel structure in the olfactory lamina propria of Efemp1(-/-) mice. Overall, these data suggest a role for fibulin-3 in tissue maintenance and regeneration in the adult olfactory pathway.

  View details for DOI 10.1016/j.matbio.2009.06.001

  View details for Web of Science ID 000272102000005

  View details for PubMedID 19520159

• The Glycoprotein Fibulin-3 Regulates Morphology and Motility of Olfactory Ensheathing Cells In Vitro GLIA Vukovic, J., Ruitenberg, M. J., Roet, K., Franssen, E., Arulpragasam, A., Sasaki, T., Verhaagen, J., Harvey, A. R., Busfield, S. J., Plant, G. W. 2009; 57 (4): 424-443

  Abstract

  The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. A specialized glial cell within the olfactory nerve, called olfactory ensheathing cell (OEC), and their associated extracellular matrix are thought to play an important role during regenerative events in this system. To gain insight into novel molecules that could mediate the OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments were conducted which revealed high expression of the glycoprotein fibulin-3 in OECs. This observation was confirmed with quantitative PCR. In vivo, the distribution of all members of the fibulin family, fibulin-3 included, was localized to the lamina propria underneath the olfactory epithelium, in close association within olfactory nerve bundles. To manipulate fibulin-3 gene expression in cultured OECs, lentiviral vector constructs were designed to either transgenically express or knock-down fibulin-3. Experimental data showed that increased levels of fibulin-3 induced profound morphological changes in cultured OECs, impeded with their migratory abilities and also suppressed OEC-mediated neurite outgrowth. Knock-down of fibulin-3 levels resulted in reduced OEC proliferation. In conclusion, the data provide novel insights into a putative role for fibulin-3 in the regulation of cell migration and neurite outgrowth within the primary olfactory pathway.

  View details for DOI 10.1002/glia.20771

  View details for Web of Science ID 000262904000007

  View details for PubMedID 18803302

• Use of GFP to analyze morphology, connectivity, and function of cells in the central nervous system. Methods in molecular biology (Clifton, N.J.) Harvey, A. R., Ehlert, E., de Wit, J., Drummond, E. S., Pollett, M. A., Ruitenberg, M., Plant, G. W., Verhaagen, J., Levelt, C. N. 2009; 515: 63-95

  Abstract

  We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with "floxed" transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.

  View details for DOI 10.1007/978-1-59745-559-6_5

  View details for PubMedID 19378117

• CX3CL1/fractalkine regulates branching and migration of monocyte-derived cells in the mouse olfactory epithelium JOURNAL OF NEUROIMMUNOLOGY Ruitenberg, M. J., Vukovic, J., Blomster, L., Hall, J. M., Jung, S., Filgueira, L., Mcmenamin, P. G., Plant, G. W. 2008; 205 (1-2): 80-85

  Abstract

  The olfactory epithelium (OE) is a site of massive adult neurogenesis where olfactory sensory neurons (OSNs) are continuously turned over. Tissue macrophages have been implicated in phagocytosis of degenerating cells but the molecular mechanisms that allow for their recruitment while maintaining a neurogenic microenvironment are poorly understood. This study reports that the neuroprotective chemokine CX3CL1 is expressed by OSNs and olfactory ensheathing cells. Monocyte-derived cells in the OE depend on CX3CL1-signalling for intraepithelial migration and apical dendrite expression. These observations are first to demonstrate phenotypic differences in appearance and distribution of monocyte-derived cells in nervous tissue due to CX3CR1 deficiency.

  View details for DOI 10.1016/j.jneuroim.2008.09.010

  View details for Web of Science ID 000261902200013

  View details for PubMedID 18951638

• The importance of transgene and cell type on the regeneration of adult retinal ganglion cell axons within reconstituted bridging grafts EXPERIMENTAL NEUROLOGY Hu, Y., Arulpragasam, A., Plant, G. W., Hendriks, W. T., Cui, Q., Harvey, A. R. 2007; 207 (2): 314-328

  Abstract

  When grafted onto the cut optic nerve, chimeric peripheral nerve (PN) sheaths reconstituted with adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Regrowth can be further enhanced by using PN containing SCs transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of ciliary neurotrophic factor (CNTF). To determine whether other neurotrophic factors or different cell types also enhance RGC regrowth in this bridging model, we tested the effectiveness of (1) adult SCs transduced with brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), and (2) fibroblasts (FBs) genetically modified to express CNTF. SCs transduced with LV-BDNF and LV-GDNF secreted measurable and bioactive amounts of each of these proteins, but reconstituted grafts containing LV-BDNF or LV-GDNF transduced SCs did not enhance RGC survival or axonal regrowth. LV-BDNF modified grafts did, however, contain many pan-neurofilament immunolabeled axons, many of which were also immunoreactive for calcitonin gene-related peptide (CGRP) and were presumably of peripheral sensory origin. Nor-adrenergic and cholinergic axons were also seen in these grafts. There were far fewer axons in LV-GDNF engineered grafts. Reconstituted PN sheaths containing FBs that had been modified to express CNTF did not promote RGC viability or regeneration, and PN reconstituted with a mixed population of SCs and CNTF expressing FBs were less effective than SCs alone. These data show that both the type of neurotrophic factor and the cell types that express these factors are crucial elements when designing bridging substrates to promote long-distance regeneration in the injured CNS.

  View details for DOI 10.1016/j.expneurol.2007.07.001

  View details for Web of Science ID 000250076700014

  View details for PubMedID 17689533

• Culture conditions affect proliferative responsiveness of olfactory ensheathing glia to neuregulins GLIA De Mello, T. R., Busfield, S., Dunlop, S. A., Plant, G. W. 2007; 55 (7): 734-745

  Abstract

  Olfactory ensheathing glia (OEG) have been used to improve outcome after experimental spinal cord injury and are being trialed clinically. Their rapid proliferation in vitro is essential to optimize clinical application, with neuregulins (NRG) being potential mitogens. We examined the effects of NRG-1beta, NRG-2alpha, and NRG3 on proliferation of p75-immunopurified adult OEG. OEG were grown in serum-containing medium with added bovine pituitary extract and forskolin (added mitogens) or in serum-containing medium (no added mitogens). Cultures were switched to chemically defined medium (no added mitogens or serum), NRG added and OEG proliferation assayed using BrdU. OEG grown initially with added mitogens were not responsive to added NRGs and pre-exposure to forskolin and pituitary extract increased basal proliferation rates so that OEG no longer responded to added NRG. However, NRG promoted proliferation but only if cells were initially grown in mitogen-free medium. Primary OEG express ErbB2, ErbB3, and small levels of ErbB4 receptors; functional blocking indicates that ErbB2 and ErbB3 are the main NRG receptors utilized in the presence of NRG-1beta. The long-term stimulation of OEG proliferation by initial culture conditions raises the possibility of manipulating OEG before therapeutic transplantation.

  View details for DOI 10.1002/glia.20502

  View details for Web of Science ID 000245679300007

  View details for PubMedID 17348025

• The chemokine receptorCX(3)CR1 mediates homing of MHC class II - Positive cells to the normal mouse corneal epithelium INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Chinnery, H. R., Ruitenberg, M. J., Plant, G. W., Pearlman, E., Jung, S., McMenamin, P. G. 2007; 48 (4): 1568-1574

  Abstract

  Recent investigations have revealed that populations of macrophages and dendritic cells (DCs) are present in the stroma and epithelium of the cornea, although the precise phenotype and distribution are still controversial. CX(3)CR1, the sole receptor for the chemokine fractalkine, is expressed by these monocyte-derived cells. Transgenic CX(3)CR1(GFP) mice, in which either one (heterozygous) or both (homozygous) copies of the CX(3)CR1 gene were replaced by enhanced green fluorescent protein (eGFP), were used to characterize monocyte-derived cells in the mouse cornea and to determine whether the expression of this receptor influences the recruitment of these cells into the normal cornea.Wholemount corneas were immunostained with anti-leukocyte antibodies to the phenotypic markers major histocompatibility complex (MHC) class II, CD169, CD68, CD11b, and CD45 and analyzed by epifluorescence and confocal microscopy. The density of intraepithelial MHC class II(+) cells was quantified in wild-type, CX(3)CR1(+/GFP) heterozygous, CX(3)CR1(GFP/GFP) homozygous, and CX(3)CR1-knockout mice.There was a significant reduction in the number of MHC class II(+) cells (putative DCs) in the corneal epithelium of CX(3)CR1-deficient mice (P < 0.009) compared with wild-type mice, and the few cells that were present did not possess classic dendriform morphology. No GFP(+) MHC class II(-) cells were noted in the epithelium. Dual immunostaining of corneas in both heterozygous and homozygous (CX(3)CR1-deficient) mice revealed GFP(+) cells with a more pleomorphic morphology throughout the entire corneal stroma that were CD11b(+) CD169(+), and had variable degrees of expression of CD68 andMHC class II. The immunophenotype and morphology of these intrastromal cells is strongly indicative of a macrophage phenotype.This study has identified a role for CX(3)CR1 in the normal recruitment of MHC class II(+) putative DCs into the corneal epithelium and establishes a model for investigating monocyte-derived cells and fractalkine/CX(3)CR1 interactions during corneal disease.

  View details for DOI 10.1167/iovs.06-0746

  View details for Web of Science ID 000245408200019

  View details for PubMedID 17389486

• Influence of adult Schwann cells and olfactory ensheathing glia on axon-target cell interactions in the CNS: a comparative analysis using a retinotectal co-graft model NEURON GLIA BIOLOGY Vukovic, J., Plant, G. W., Ruitenberg, M. J., Harvey, A. R. 2007; 3: 105-117

  Abstract

  We used an in vivo transplant approach to examine how adult Schwann cells and olfactory ensheathing glia OEG influence the specificity of axon-target cell interactions when they are introduced into the CNS. Populations of either Schwann cells or OEG were mixed with dissociated fetal tectal cells presumptive superior colliculus and, after reaggregation, pieces were grafted onto newborn rat superior colliculus. Both glial types were prelabeled with lentiviral vectors encoding green fluorescent protein. Grafts rapidly established fiber connections with the host and retinal projections into co-grafts were assessed 656 days posttransplantation by injecting cholera toxin B into host eyes. In control rats that received pure dissociated-reaggregated tectal grafts, retinal ganglion cell RGC axons selectively innervated defined target areas, corresponding to the retinorecipient layer in normal superior colliculus. The pattern of RGC axon ingrowth into OEG containing co-grafts was similar to that in control grafts. However, in Schwann cell co-grafts there was reduced host retinal input into presumptive target areas and many RGC axons were scattered throughout the neuropil. Given that OEG in co-grafts had minimal impact on axon-target cell recognition, OEG might be an appropriate cell type for direct transplantation into injured neuropil when attempting to stimulate specific pathway reconstruction.

  View details for DOI 10.1017/S1740925X07000671

  View details for Web of Science ID 000254434700003

  View details for PubMedID 18634570

• AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells GENE THERAPY Leaver, S. G., Cui, Q., Plant, G. W., Arulpragasam, A., Hisheh, S., Verhaagen, J., Harvey, A. R. 2006; 13 (18): 1328-1341

  Abstract

  We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

  View details for Web of Science ID 000240237900003

  View details for PubMedID 16708079

• Gene therapy and transplantation in CNS repair: The visual system PROGRESS IN RETINAL AND EYE RESEARCH Harvey, A. R., Hu, Y., Leaver, S. G., Mellough, C. B., Park, K., Verhaagen, J., Plant, G. W., Cui, Q. 2006; 25 (5): 449-489

  Abstract

  Normal visual function in humans is compromised by a range of inherited and acquired degenerative conditions, many of which affect photoreceptors and/or retinal pigment epithelium. As a consequence the majority of experimental gene- and cell-based therapies are aimed at rescuing or replacing these cells. We provide a brief overview of these studies, but the major focus of this review is on the inner retina, in particular how gene therapy and transplantation can improve the viability and regenerative capacity of retinal ganglion cells (RGCs). Such studies are relevant to the development of new treatments for ocular conditions that cause RGC loss or dysfunction, for example glaucoma, diabetes, ischaemia, and various inflammatory and neurodegenerative diseases. However, RGCs and associated central visual pathways also serve as an excellent experimental model of the adult central nervous system (CNS) in which it is possible to study the molecular and cellular mechanisms associated with neuroprotection and axonal regeneration after neurotrauma. In this review we present the current state of knowledge pertaining to RGC responses to injury, neurotrophic and gene therapy strategies aimed at promoting RGC survival, and how best to promote the regeneration of RGC axons after optic nerve or optic tract injury. We also describe transplantation methods being used in attempts to replace lost RGCs or encourage the regrowth of RGC axons back into visual centres in the brain via peripheral nerve bridges. Cooperative approaches including novel combinations of transplantation, gene therapy and pharmacotherapy are discussed. Finally, we consider a number of caveats and future directions, such as problems associated with compensatory sprouting and the reformation of visuotopic maps, the need to develop efficient, regulatable viral vectors, and the need to develop different but sequential strategies that target the cell body and/or the growth cone at appropriate times during the repair process.

  View details for DOI 10.1016/j.preteyeres.2006.07.002

  View details for Web of Science ID 000241967200002

  View details for PubMedID 16963308

• Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro GLIA Leaver, S. G., Harvey, A. R., Plant, G. W. 2006; 53 (5): 467-476

  Abstract

  In vivo, transplanted adult olfactory ensheathing glia (OEG) and adult Schwann cells (SC) can support the regrowth of at least some transected axons within adult CNS neuropil. In the present study, we developed an in vitro adult rat retinal explant model to explore the influence of primary adult SC and OEG on retinal ganglion cell (RGC) neurite regrowth in the presence of glial cells endogenous to the retina. Retinal quadrants were plated RGC-side down onto aclar hats coated with either pure collagen (type 1), collagen with OEG, collagen with SCs, or collagen coated with both OEG and SCs. Regrowing retinal neurites extended onto the pure collagen substrate, largely in association with astrocytes that migrated out from the explants (mean number of neurites: 144+/-65 SEM). The additional presence of OEG (669+/-122), but not SCs (97+/-41), supported the regrowth of significantly greater numbers of RGC neurites. Furthermore, this OEG-stimulated regeneration was over significantly greater distances; >68% of neurites extended >500 microm from the explant, compared with explants plated onto SCs or collagen alone (15% and 29%, respectively). When OEG and SCs were co-cultured the number of regenerating neurites was reduced (397+/-81) compared with the pure OEG treatment. Analysis of explants on pure collagen substrates fed with media conditioned by purified OEG or SC showed no increase in neurite outgrowth compared with control treatments, suggesting that the enhanced growth in the presence of OEG is a contact-mediated effect. The observed differences between the abilities of OEG and SC to support the growth of CNS-derived fibers in the presence of astrocytes support the suggestion that OEG may be better suited for direct transplantation into CNS neuropil following injury.

  View details for DOI 10.1002/glia.20311

  View details for Web of Science ID 000235611900002

  View details for PubMedID 16355371

• Olfactory ensheathing cells: Characteristics, genetic engineering, and therapeutic potential JOURNAL OF NEUROTRAUMA Ruitenberg, M. J., Vukovic, J., Sarich, J., Busfield, S. J., Plant, G. W. 2006; 23 (3-4): 468-478

  Abstract

  Injured neurons in the mammalian central nervous system (CNS) do not normally regenerate their axons after injury. Neurotrauma to the CNS usually results in axonal damage and subsequent loss of communication between neuronal networks, causing long-term functional deficits. For CNS regeneration, repair strategies need to be developed that promote regrowth of lesioned axon projections and restoration of neuronal connectivity. After spinal cord injury (SCI), cystic cavitations are often found, particularly in the later stages, due to the loss of neural tissue at the original impact site. Ultimately, for the promotion of axonal regrowth in these situations, some form of transplantation will be required to provide lesioned axons with a supportive substrate along which they can extend. Here, we review the use of olfactory ensheathing cells: their location and role in the olfactory system, their use as cellular transplants in SCI paradigms, alone or in combination with gene therapy, and the unique properties of these cells that may give them a potential advantage over other cellular transplants.

  View details for Web of Science ID 000237337700018

  View details for PubMedID 16629630

• Lentiviral-mediated transfer of CNTF to Schwann cells within reconstructed peripheral nerve grafts enhances adult retinal ganglion cell survival and axonal regeneration MOLECULAR THERAPY Hu, Y., Leaver, S. G., Plant, G. W., Hendriks, W. T., Niclou, S. P., Verhaagen, J., Harvey, A. R., Cui, Q. 2005; 11 (6): 906-915

  Abstract

  We recently described a method for reconstituting peripheral nerve (PN) sheaths using adult Schwann cells (SCs). Reconstructed PN tissue grafted onto the cut optic nerve supports the regeneration of injured adult rat retinal ganglion cell (RGC) axons. To determine whether genetic manipulation of such grafts can further enhance regeneration, adult SCs were transduced with lentiviral vectors encoding either ciliary neurotrophic factor (LV-CNTF) or green fluorescent protein (LV-GFP). SCs expressed transgenes for at least 4 weeks after transplantation. There were high levels of CNTF mRNA and CNTF protein in PN grafts containing LV-CNTF-transduced SCs. Mean RGC survival was significantly increased with these grafts (11,863/retina) compared with LV-GFP controls (7064/retina). LV-CNTF-transduced SCs enhanced axonal regeneration to an even greater extent (3097 vs 393 RGCs/retina in LV-GFP controls). Many regenerated axons were myelinated. The use of genetically modified, reconstituted PN grafts to bridge tissue defects may provide new therapeutic strategies for the treatment of both CNS and PNS injuries.

  View details for DOI 10.1016/j.ymthe.2005.01.016

  View details for Web of Science ID 000229732700014

  View details for PubMedID 15922961

• NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration BRAIN Ruitenberg, M. J., Levison, D. B., Lee, S. V., Verhaagen, J., Harvey, A. R., Plant, G. W. 2005; 128: 839-853

  Abstract

  Adenoviral (AdV) vectors encoding neurotrophin-3 (AdV-NT-3) or the bacterial marker enzyme beta-galactosidase (LacZ gene) were used to transduce olfactory ensheathing glia (OEG) cultures. AdV vector-transduced OEG expressed high levels of recombinant neurotrophin as shown by in situ hybridization and enzyme-linked immunosorbent assay techniques. The biological activity of vector-derived NT-3 was determined in a dorsal root ganglia neurite outgrowth assay. Engineered cell suspensions were then injected into adult Fischer 344 rat spinal cord immediately after unilateral cervical (C4) corticospinal tract (CST) transection. Transplanted animals received a total of 200,000 cells; either non-transduced OEG or OEG transduced with AdV vectors encoding NT-3 or LacZ, respectively. At 3 months after injury, lesion volumes were significantly smaller in all OEG-transplanted rats when compared with control (medium-injected) rats. Anterograde tracing of the lesioned CST projection, originating from the contralateral sensorimotor cortex, showed a significantly greater number of distal CST axons only in OEG-NT-3-transplanted rats. Behavioural analysis was performed on all rats using open field locomotion scoring, and a forelimb reaching task with Eshkol-Wachman movement notation. Analysis of behavioural tests revealed no significant differences in recovery between experimental groups, although movement analysis indicated that possible compensatory mechanisms were occurring after OEG implantation. The results demonstrate that OEG transplantation per se can promote tissue sparing after injury, but, after appropriate genetic modification, these olfactory-derived cells become far more effective in promoting long-distance maintenance/regeneration of lesioned adult CST axons.

  View details for DOI 10.1093/brain/awh424

  View details for Web of Science ID 000227977200020

  View details for PubMedID 15716305

• Ex vivo adenoviral vector-mediated neurotrophin gene transfer to olfactory ensheathing glia: Effects on rubrospinal tract regeneration, lesion size, and functional recovery after implantation in the injured rat spinal cord JOURNAL OF NEUROSCIENCE Ruitenberg, M. J., Plant, G. W., Hamers, F. P., Wortel, J., Blits, B., Dijkhuizen, P. A., Gispen, W. H., Boer, G. J., Verhaagen, J. 2003; 23 (18): 7045-7058

  Abstract

  The present study uniquely combines olfactory ensheathing glia (OEG) implantation with ex vivo adenoviral (AdV) vector-based neurotrophin gene therapy in an attempt to enhance regeneration after cervical spinal cord injury. Primary OEG were transduced with AdV vectors encoding rat brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or bacterial marker protein beta-galactosidase (LacZ) and subsequently implanted into adult Fischer rats directly after unilateral transection of the dorsolateral funiculus. Implanted animals received a total of 2 x 105 OEG that were subjected to transduction with neurotrophin-encoding AdV vector, AdV-LacZ, or no vector, respectively. At 4 months after injury, lesion volumes were smaller in all OEG implanted rats and significantly reduced in size after implantation of neurotrophin-encoding AdV vector-transduced OEG. All OEG grafts were filled with neurofilament-positive axons, and AdV vector-mediated expression of BDNF by implanted cells significantly enhanced regenerative sprouting of the rubrospinal tract. Behavioral analysis revealed that OEG-implanted rats displayed better locomotion during horizontal rope walking than unimplanted lesioned controls. Recovery of hind limb function was also improved after implantation of OEG that were transduced with a BDNF- or NT-3-encoding AdV vector. Hind limb performance during horizontal rope locomotion did directly correlate with lesion size, suggesting that neuroprotective effects of OEG implants contributed to the level of functional recovery. Thus, our results demonstrate that genetic engineering of OEG not only resulted in a cell that was more effective in promoting axonal outgrowth but could also lead to enhanced recovery after injury, possibly by sparing of spinal tissue.

  View details for Web of Science ID 000184587100009

  View details for PubMedID 12904465

• Delayed transplantation of olfactory ensheathing glia promotes sparing/regeneration of supraspinal axons in the contused adult rat spinal cord JOURNAL OF NEUROTRAUMA Plant, G. W., Christensen, C. L., Oudega, M., Bunge, M. B. 2003; 20 (1): 1-16

  Abstract

  The aim of this study was to determine the preferred time and environment for transplantation of olfactory ensheathing glia (OEG) into the moderately contused adult rat thoracic spinal cord. Purified OEG were suspended in culture medium with or without fibrinogen and injected into the contused cord segment at 30 min or 7 days after injury. Control animals received a contusion injury only or injection of only medium 7 days after contusion. The effects on axonal sparing/regeneration and functional recovery were evaluated 8 weeks after injury. The grafts largely filled the lesion site, reducing cavitation, and appeared continuous with the spinal nervous tissue. Whereas in 7d/medium only animals, 54% of spinal tissue within a 2.5-mm-long segment of cord centered at the injury site was spared, significantly more tissue was spared in 0 d/OEG-medium (73%), 0 d/OEG-fibrin (66%), 7 d/OEG-medium (70%), and 7 d/OEG-fibrin (68%) grafted animals. Compared with controls, the grafted animals exhibited more serotonergic axons within the transplant, the surrounding white matter, and the spinal cord up to at least 20 mm caudal to the graft. Retrograde tracing revealed that all but the 0 d/OEG-fibrin graft promoted sparing/regeneration of supraspinal axons compared with controls. Overall, the 7 d/OEG-medium group resulted in the best response, with twice as many labeled neurons in the brain compared with 7 d/medium only controls. Of the labeled neurons, 68% were located in the reticular formation, and 4% in the red, 4% in the raphe, and 5% in the vestibular nuclei. Hindlimb performance was modestly but significantly improved in the 7 d/OEG-medium group. Our results demonstrate that transplantation of OEG into the moderately contused adult rat thoracic spinal cord promotes sparing/regeneration of supraspinal axons and that 7 d transplantation is more effective than acute transplantation of OEG. Our results have relevant implications for future surgical repair strategies of the contused spinal cord.

  View details for Web of Science ID 000180723200001

  View details for PubMedID 12614584

• A new approach to CNS repair using chimeric peripheral nerve grafts JOURNAL OF NEUROTRAUMA Cui, Q., Pollett, M. A., Symons, N. A., Plant, G. W., Harvey, A. R. 2003; 20 (1): 17-31

  Abstract

  We have examined whether transplanted freeze-thawed peripheral nerve (PN) sheaths repopulated ex vivo with purified adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Cultured adult SCs were derived from donor rats or from the host animals themselves. We also transplanted PN sheaths filled with neonatal SCs or donor adult olfactory ensheathing glia (OEG). 100,000 cells were injected into 1.5-cm lengths of freeze-thawed PN. After 2 days in culture, repopulated PN segments were grafted onto the transected optic nerve of adult Fischer rats. Three weeks later, 6% fluorogold (FG) was applied to distal PN. Retrogradely labeled RGCs were counted in retinal wholemounts and PN grafts were processed for immunohistochemistry. As expected, there was no RGC axon regeneration in cell-free grafts. Regrowth was also absent in neonatal SC- and adult OEG-filled grafts, which contained only small numbers of surviving donor cells. Many cells were, however, seen in adult SC repopulated PN grafts, intermingled with pan-neurofilament(+) and GAP-43(+) fibers. SCs were aligned along the grafts and were S-100(+), p75(+). Ultrastructurally, SCs were associated with myelinated and unmyelinated axons. Hundreds of FG-labeled RGCs were seen in retinas of rats with congeneic or allogeneic PN sheaths repopulated with either donor or autologous (host-derived) adult SCs. Intraocular CNTF injections significantly increased the number of regenerating RGCs in donor and autologous adult SC groups. The use of chimeric grafts to bridge CNS tissue defects could provide a clinical alternative to using multiple PN autografts, the harvesting of which would exacerbate peripheral dysfunction in already injured patients.

  View details for Web of Science ID 000180723200002

  View details for PubMedID 12614585

• Purified adult ensheathing glia fail to myelinate axons under culture conditions that enable Schwann cells to form myelin JOURNAL OF NEUROSCIENCE Plant, G. W., Currier, P. F., Cuervo, E. P., Bates, M. L., Pressman, Y., Bunge, M. B., Wood, P. M. 2002; 22 (14): 6083-6091

  Abstract

  Several studies have suggested that olfactory ensheathing glia (EG) can form Schwann cell (SC)-like myelin. Because of possible misinterpretation attributable to contaminating SCs, the capacity of EG to produce myelin needs to be explored further. Therefore, we compared the abilities of adult EG, purified by immunopanning with p75 antibody, and adult SCs to produce myelin when cocultured with purified dorsal root ganglion neurons (DRGNs) in serum-free and serum-containing media. In both media formulations, the number of myelin sheaths in SC/DRGN cultures was far higher than in EG/DRGN cultures; the number of sheaths in EG/DRGN cultures was equal to that in purified DRGN cultures without added cells. The latter result demonstrates that myelination by a few SCs remaining in purified DRGN cultures may occur, suggesting that myelin in EG/DRGN cultures could be SC myelin. Striking differences in the relationship of EG and SC processes to axons were observed. Whereas SCs displayed relatively short, thick processes that engulfed axons in small bundles or in individual cytoplasmic furrows and segregated larger axons into one-to-one relationships, EG extended flattened sheets that partitioned or only partially encircled fascicles of axons, sometimes spanning the entire culture. SCs exhibited behavior typical of SCs in peripheral nerves, whereas EG exhibited characteristics resembling those of EG in olfactory nerves. In sum, p75-selected EG from adult animals did not exhibit an SC-like relationship to axons and did not form myelin.

  View details for Web of Science ID 000176840500032

  View details for PubMedID 12122069

• Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord GENE THERAPY Ruitenberg, M. J., Plant, G. W., Christensen, C. L., Blits, B., Niclou, S. P., Harvey, A. R., Boer, G. J., Verhaagen, J. 2002; 9 (2): 135-146

  Abstract

  Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal 'bridging' substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.

  View details for DOI 10.1038/sj/gt/3301626

  View details for Web of Science ID 000173886200007

  View details for PubMedID 11857072

• Mitogenic response of adult rat olfactory ensheathing glia to four growth factors GLIA Yan, H., Bunge, M. B., Wood, P. M., Plant, G. W. 2001; 33 (4): 334-342

  Abstract

  Olfactory ensheathing glia (EG) from adult rat proliferate slowly in vitro without added mitogens. The potential future use of EG in transplantation within the central nervous system to improve neural repair is dependent on identifying mitogens that will effectively expand EG without altering their phenotype. The mitogenic effects of heregulin (HRG), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), and forskolin (FSK) on cultured adult-derived rat EG were monitored by tritiated-thymidine labeling and p75 immunostaining. In serum-containing medium, HRG, FGF-2, PDGF-BB, IGF-1, and FSK were capable of stimulating EG proliferation, and the stimulation by these growth factors was potentiated by FSK. The combinations of HRG + FGF-2, HRG + PDGF-BB, HRG + IGF-1, FGF-2 + PDGF-BB, and FGF-2 + IGF-1 all promoted EG proliferation in an additive manner. In serum-free medium, HRG and FGF-2 were mitogenic, but PDGF-BB, IGF-1 and FSK were not; however, FSK potentiated the stimulation by HRG and FGF-2, and the combination of HRG + FGF-2 promoted EG proliferation in an additive manner. This new information will be useful for the design of protocols to achieve sufficient numbers of adult-derived EG for clinical purposes. This study also further establishes similarities between EG and Schwann cells.

  View details for Web of Science ID 000167654700008

  View details for PubMedID 11246232

• Inhibitory proteoglycan immunoreactivity is higher at the caudal than the rostral Schwann cell graft-transected spinal cord interface MOLECULAR AND CELLULAR NEUROSCIENCE Plant, G. W., Bates, M. L., Bunge, M. B. 2001; 17 (3): 471-487

  Abstract

  To begin to evaluate the influence that proteoglycans may have on the success of Schwann cell (SC) transplants to induce axonal regrowth across a complete transection lesion and beyond, we determined the pattern of expression of inhibitory chondroitin sulfate proteoglycans (CSPGs) 3 weeks after transplantation into completely transected adult rat thoracic spinal cord. Using immunohistochemistry, we observed that: (1) CSPGs recognized by CS-56 antibody are present on astrocytes, fibroblasts, and SCs in the distal graft, and at lesion and cystic cavity borders; (2) CS-56 immunoreactivity (IR) is greater at the caudal SC graft-host cord interface than the rostral interface; (3) phosphacan-IR, also greater at the caudal interface, is associated with astrocytes, fibroblasts, as yet unidentified cells, and extracellular matrix; (4) neurocan-IR is present on astrocytes and as yet unidentified cells in grey and white matter; and (5) NG2-IR is associated with matrix near SC grafts, unidentified cells mainly in white matter, and lesion borders and cysts. Neither oligodendrocytes nor activated macrophages/microglia were immunostained. In sum, the CSPGs studied are increased at 3 weeks, especially at the caudal SC graft-cord interface, possibly contributing to an inhibitory molecular barrier that precludes regrowing descending axons from entering the caudal host cord.

  View details for DOI 10.1006/mcne.2000.0948

  View details for Web of Science ID 000167832000005

  View details for PubMedID 11273643

• A new type of biocompatible bridging structure supports axon regrowth after implantation into the lesioned rat optic tract CELL TRANSPLANTATION Plant, G. W., Harvey, A. R. 2000; 9 (6): 759-772

  Abstract

  We have developed a new type of polymer/cell/matrix implant and tested whether it can promote the regrowth of retinal ganglion cell (RGC) and other axons across surgically induced tissue defects in the CNS. The constructs, which consisted of 2-2.5-mm-long polycarbonate tubes filled with lens capsule-derived extracellular matrix coated with cultured neonatal Schwann cells, were implanted into lesion cavities made in the left optic tract (OT) of 18-21-day-old rats. In one group, to promote Schwann cell proliferation and perhaps also to stimulate axon regrowth, basic fibroblast growth factor (bFGF) was added to the lens capsule matrix prior to implantation. In another group, to determine whether application of growth factors to the somata of cells enhances the regrowth of distally injured axons, the neurotrophin NT-4/5 was injected into the eye contralateral to the OT lesion. NT-4/5 and bFGF treatments were combined in some rats. After medium-term (4-10 weeks) or long-term (15-20 weeks) survivals, axon growth into implants was assessed immunohistochemically using a neurofilament (RT97) antibody. RGC axons were visualized after injection of WGA/HRP into the right eye. Viable Schwann cells were present in implants at all times after transplantation. Large numbers of RT97+ axons were consistently found within the bridging implants, often associated with the peripheral glia. Axons were traced up to 1.7 mm from the nearest CNS neuropil and there was immunohistochemical evidence of myelination by Schwann cells and by host oligodendrocytes. There were fewer RGC axons in the implants, fibers growing up to 1.6 mm from the thalamus. Neither NT-4/5 nor bFGF, alone or in combination, significantly increased the extent of RGC axon growth within the implants. A group of OT-lesioned rats was implanted with polymer tubes filled with 2-2.5-mm-long pieces of predegenerate peripheral nerve. Surprisingly, polymer/cell/matrix constructs contained comparatively greater numbers of RGC and other axons and supported more extensive axon elongation. Thus, implants of this type may potentially be useful in bridging large tissue defects in the CNS.

  View details for Web of Science ID 000166532900003

  View details for PubMedID 11202563

• Poly(alpha-hydroxyacids) for application in the spinal cord: Resorbability and biocompatibility with adult rat Schwann cells and spinal cord JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Gautier, S. E., Oudega, M., Fragoso, M., Chapon, P., Plant, G. W., Bunge, M. B., Parel, J. M. 1998; 42 (4): 642-654

  Abstract

  Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.

  View details for Web of Science ID 000076795500022

  View details for PubMedID 9827690

• Implantation of collagen IV Poly(2-hydroxyethyl methacrylate) hydrogels containing schwann cells into the lesioned rat optic tract CELL TRANSPLANTATION Plant, G. W., Chirila, T. V., Harvey, A. R. 1998; 7 (4): 381-391

  Abstract

  Poly (2-hydroxyethylmethacrylate) (PolyHEMA) hydrogels, when combined with extracellular matrix molecules and infiltrated with cultured Schwann cells, have the capability to induce CNS axonal regrowth after injury. We have further investigated these PolyHEMA hydrogels and their potential to bridge CNS injury sites. Collagen IV-impregnated hydrogels containing Schwann cells were implanted into the lesioned optic tract in 14 rats. On examination 2-4 months later, there was good adherence between the implants and CNS tissue, and large numbers of viable Schwann cells (S100+, GFAP+, Laminin+, and LNGFR+) were seen within the hydrogel matrices. Immunohistochemical analysis showed that the collagen IV-impregnated PolyHEMA hydrogels preferentially supported the transplanted Schwann cells and not host glial cells such as astrocytes (GFAP+) or oligodendroglia (CAII+). Macrophages (ED1+) were also seen within the sponge structure. Eighty-three percent of the implanted hydrogels contained RT97+ axons within their trabecular networks. Regrowing axons were associated with the transplanted Schwann cells and not with the small number of infiltrating astrocytes. RT97+ axons were traced up to 510 microm from the nearest host neuropil. These axons were sometimes myelinated by the transplanted Schwann cells and expressed the peripheral myelin marker Po+. WGA/HRP-labeled retinal axons were seen within transplanted hydrogel sponges, with 40% of the cases growing for distances up to 350-450 microm within the polymer network. The data indicate that impregnating PolyHEMA sponges with collagen IV can modify the host glial reaction and support the survival of transplanted Schwann cells. This study thus provides new information on how biomaterials could be used to modify and bridge CNS injury sites.

  View details for Web of Science ID 000075039500006

  View details for PubMedID 9710307

• Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants JOURNAL OF NEUROSCIENCE Ramon-Cueto, A., Plant, G. W., Avila, J., Bunge, M. B. 1998; 18 (10): 3803-3815

  Abstract

  The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional impairment. To induce axonal regeneration in the transected adult rat spinal cord, we have used the axonal growth-promoting properties of adult olfactory bulb ensheathing glia (EG). Schwann cell (SC)-filled guidance channels were grafted to bridge both cord stumps, and suspensions of pure (98%) Hoechst-labeled EG were stereotaxically injected into the midline of both stumps, 1 mm from the edges of the channel. In EG-transplanted animals, numerous neurofilament-, GAP-43-, anti-calcitonin gene-related peptide (CGRP)-, and serotonin-immunoreactive fibers traversed the glial scars formed at both cord-graft interfaces. Supraspinal serotonergic axons crossed the transection gap through connective tissue bridges formed on the exterior of the channels, avoiding the channel interior. Strikingly, after crossing the distal glial scar, these fibers elongated in white and periaqueductal gray matter, reaching the farthest distance analyzed (1.5 cm). Tracer-labeled axons present in SC grafts were found to extend across the distal interface and up to 800 microm beyond in the distal cord. Long-distance regeneration (at least 2.5 cm) of injured ascending propriospinal axons was observed in the rostral spinal cord. Transplanted EG migrated longitudinally and laterally from the injection sites, reaching the farthest distance analyzed (1.5 cm). They moved through white matter tracts, gray matter, and glial scars, overcoming the inhibitory nature of the CNS environment, and invaded SC and connective tissue bridges and the dorsal and ventral roots adjacent to the transection site. Transplanted EG and regenerating axons were found in the same locations. Because EG seem to provide injured spinal axons with appropriate factors for long-distance elongation, these cells offer new possibilities for treatment of CNS conditions that require axonal regeneration.

  View details for Web of Science ID 000073484300031

  View details for PubMedID 9570810

• In vitro assessment of the biological activity of basic fibroblast growth factor released from various polymers and biomatrices JOURNAL OF BIOMATERIALS APPLICATIONS Davies, M. J., Mitchell, C. A., Maley, M. A., Grounds, M. D., Harvey, A. R., Plant, G. W., Wood, D. J., Hong, Y., Chirila, T. V. 1997; 12 (1): 31-56

  Abstract

  The kinetics of controlled release of basic fibroblast growth factor (bFGF) from polymers (sutures, polycarbonate, Hydron, and Elvax), biopolymers (alginate), and biomatrices (lens capsules), and conditions for storage of bFGF (temperature, plastic type, heparin) were evaluated in vitro. Tissue culture proliferation bioassays with 3T3 fibroblasts, showed that only lens capsules with bFGF had a sustained release of bFGF for up to three weeks. The other materials released all of the 'bound' bFGF with two hours or produced an inflammatory response in vivo. Therefore, the lens tissue had the most potential for controlled long-term delivery of bFGF in vivo. These studies emphasise the importance of in vitro analysis of release kinetics of growth factors from a range of materials as a basis for potential in vivo applications.

  View details for Web of Science ID A1997XU09100002

  View details for PubMedID 9285329

• Hydrogels containing peptide or aminosugar sequences implanted into the rat brain: Influence on cellular migration and axonal growth EXPERIMENTAL NEUROLOGY Plant, G. W., Woerly, S., Harvey, A. R. 1997; 143 (2): 287-299

  Abstract

  Biocompatible polymer matrices for implantation into lesion sites in the brain were synthesized by incorporating peptide or aminosugar sequences into N-(2-hydroxypropyl)methacrylamide (HPMA) hydrogels. RGD peptide sequences were chemically linked to the hydrogel backbone via a glycylglycine spacer; aminosugars were glucosamine (NHGlc) or N-acetylglucosamine residues. Unmodified or sequence containing HPMA hydrogels were implanted into the lesioned optic tract or cerebral cortex of juvenile (17- to 19-day-old) or adult rat brains, respectively. After 10-12 months host animals were perfused and the brains were processed for immunohistochemistry using antibodies to neurofilaments (RT97), laminin, glial fibrillary acidic protein (GFAP), carbonic anhydrase II (CAII), S100 protein, macrophages (ED1), and myelin basic protein (MBP). Unmodified (control) HPMA hydrogels contained no cellular infiltration or axonal growth. Peptide (RGD)- and aminosugar-modified hydrogels showed increased adhesion properties with host neural tissue, were vascularized, and were infiltrated by host nonneuronal cells. Astrocytes (GFAP+) and macrophages (ED1(+)) were the major cell types seen within modified HPMA hydrogels, the largest numbers being found in RGD-containing polymers. CAII+ oligodendroglia were not seen within any of the hydrogel matrices. RT97(+)/MBP- axons grew into both the RGD and NHGlc hydrogel matrices for small distances. The number of axons was greatest in hydrogels implanted into cerebral cortex but in both cortex and optic tract implants the highest density of axons was seen in polymers containing RGD. The findings of this study are discussed in the context of CNS tissue replacement and the construction of bioactive scaffolds to promote regenerative axonal growth across areas of injury in the brain and spinal cord.

  View details for Web of Science ID A1997WM44900011

  View details for PubMedID 9056391

• Cultured rat neuronal and glial cells entrapped within hydrogel polymer matrices: A potential tool for neural tissue replacement NEUROSCIENCE LETTERS Woerly, S., Plant, G. W., Harvey, A. R. 1996; 205 (3): 197-201

  Abstract

  Cultured Schwann cells, neonatal astrocytes or cells dissociated from embryonic cerebral hemispheres were dispersed within poly-[N-(2-hydroxypropyl)-methacrylamide]-based hydrogel matrices by gel entrapment and maintained in vitro for 1-6 days. Glial cells were pre-labelled with Hoechst 33342. Cell differentiation and viability were studied by immunocytochemistry. Up to 15% of Schwann cells initially entrapped within the polymer matrices were immunopositive for the low affinity nerve growth factor receptor, S100, glial fibrillary acidic protein (GFAP) and laminin; up to 10% of pre-labelled astrocytes were immunopositive for GFAP and laminin. Embryonic neurons displayed immunostaining for neurofilaments. Hydrogels containing entrapped Schwann cells were implanted into the rat neocortex. These polymers supported cellular and axonal ingrowth within parts of the polymer matrix.

  View details for Web of Science ID A1996TZ59200014

  View details for PubMedID 8852592

• Neural tissue engineering: From polymer to biohybrid organs BIOMATERIALS Woerly, S., Plant, G. W., Harvey, A. R. 1996; 17 (3): 301-310

  Abstract

  This investigation reports on the immobilization of neuronal and glial cells (Schwann cells and astrocytes) within N-(2-hydroxypropyl) methacrylamide (HPMA) polymer hydrogels for the production of cell-based polymer hybrid devices. Cells were included within HPMA polymer networks by gel-entrapment, and these biogels were maintained in vitro for up to 6 days. Cell viability and differentiation were studied using immunocytochemical methods and image analysis techniques. The polymer structure and its relationships with cells were examined by scanning electron microscopy. A proportion of the cell population was viable, expressing its own antigenic profile throughout the period of gel incubation, as cells do in conventional culture conditions, and some cells exhibited behaviour such as spreading or process outgrowth and secretion of laminin. The result of the present study allows us to envisage tissue replacement in the central nervous system by means of such cell-based polymer constructs.

  View details for Web of Science ID A1996TY45900009

  View details for PubMedID 8745327

• SCHWANN-CELLS AND FETAL TECTAL TISSUE COGRAFTED TO THE MIDBRAIN OF NEWBORN RATS - FATE OF SCHWANN-CELLS AND THEIR INFLUENCE ON HOST RETINAL INNERVATION OF GRAFTS EXPERIMENTAL NEUROLOGY Harvey, A. R., Plant, G. W. 1995; 134 (2): 179-191

  Abstract

  Schwann cells transplanted into the adult central nervous system (CNS) can exert powerful growth-promoting effects on damaged axons. An important issue is whether central axons induced to regrow by Schwann cells retain the capacity to recognize and selectively innervate their appropriate target cells. To examine how Schwann cells may influence the specificity of neuron-neuron interactions in CNS neuropil, we cultured neonatal rat Schwann cells and mixed them with dissociated fetal tectal cells. In some instances, Schwann cells were prelabeled with Hoechst dye 33342. Schwann cells comprised between 2.5 and 15% of the combined cell population. After reaggregation, cografts were injected onto the midbrain of newborn rats. One to 6 months later, grafts were examined for the presence of Schwann cells and the pattern and density of host retinal innervation of the cografts was assessed. Immunohistochemical studies showed that areas of the transplants containing large numbers of surviving Hoechst-labeled Schwann cells were strongly immunoreactive for the low-affinity nerve growth factor receptor (p75), S-100, GFAP, and laminin. Very little peripheral (Po positive) myelin was seen. As in pure fetal tectal grafts, host retinal axons were sometimes observed to innervate superficial, localized areas in the cografts known to be homologous to the retinorecipient layers of the superior colliculus. Unlike pure tectal grafts, however, optic axons were not confined to these regions and fibers were often dispersed within the cograft neuropil. Dense growth was seen in association with Hoechst-labeled Schwann cells and, in some cases, optic axons were observed to grow toward Schwann cells and away from nearby target areas. These observations suggest that, under certain circumstances, Schwann cells can stimulate retinal axons to grow into inappropriate (nontarget) regions in the CNS, presumably by producing growth promoting factors which mask or compete with signals released from the target neurons themselves.

  View details for Web of Science ID A1995RX57100002

  View details for PubMedID 7556537

• SCHWANN-CELLS AND THE REGROWTH OF AXONS IN THE MAMMALIAN CNS - A REVIEW OF TRANSPLANTATION STUDIES IN THE RAT VISUAL-SYSTEM 1994 Australian-Physiological-and-Pharmacological-Society Symposium on Respiratory Systems Harvey, A. R., Plant, G. W., Tan, M. M. BLACKWELL SCIENCE PUBL AUSTR. 1995: 569–79

  Abstract

  1. We have used peripheral nerve transplants or cultured Schwann cells grafted in association with different types of polymer to study axonal regrowth in the rat visual system. In some instances the glia were co-grafted with fetal tectal tissue. 2. The studies have two main aims: (i) to determine whether retinal axons can be induced to regrow at a site distant from their cell soma, that is, after damage to the brachial region of the optic tract; (ii) to determine whether retinal axons exposed to Schwann cells retain the ability to recognize their appropriate target neurons in CNS tissue. 3. In brachial lesion studies, Schwann cells were placed in the lesion site in association with nitrocellulose papers, within polycarbonate tubes in the presence or absence of a supporting extracellular matrix (ECM), or within polymer hydrogel scaffolds. Autologous sciatic nerve grafts were also used. Immunohistochemical studies revealed the presence of regenerating axons within all polymer bridges. Regrowth of retinal axons was also seen, however, growth was not extensive and was limited to the proximal 1-1.5 mm of the implants. 4. In target innervation experiments, two surgical paradigms were developed. In one experiment, a segment of sciatic nerve was autografted onto the transected optic nerve in adult rats and the distal end of each graft was placed adjacent to fetal tectal (target) tissue implanted into the frontal cortex. To date, we have not been able to demonstrate selective recognition of target regions within tectal transplants by retinal axons exiting the sciatic nerve implants. 5. In the second experiment, Schwann cells were mixed with fetal tectal cells and co-grafted to the midbrain of newborn host rats. Schwann cells altered the characteristic pattern of host retinal growth into tectal grafts; in some cases axons were induced to grow away from appropriate target areas by nearby co-grafted Schwann cells. 6. In summary, Schwann cell/polymer scaffolds may provide a useful way of promoting the regrowth of damaged axons in the CNS, however: (i) in adults, at least, their effectiveness is reduced if they are located at a distance from the cell bodies giving rise to regenerating axons; (ii) in some circumstances exposure to a peripheral glial environment may affect the capacity of regenerating axons to recognize appropriate target cells in the CNS neuropil.

  View details for Web of Science ID A1995RR80500012

  View details for PubMedID 7586715

• AXONAL GROWTH WITHIN POLY(2-HYDROXYETHYL METHACRYLATE) SPONGES INFILTRATED WITH SCHWANN-CELLS AND IMPLANTED INTO THE LESIONED RAT OPTIC TRACT BRAIN RESEARCH Plant, G. W., Harvey, A. R., Chirila, T. V. 1995; 671 (1): 119-130

  Abstract

  Porous hydrophilic sponges made from 2-hydroxyethyl methacrylate (HEMA) have a number of possible biomedical applications. We have investigated whether these poly(HEMA) hydrogels, when coated with collagen and infiltrated in vitro with cultured Schwann cells, can be implanted into the lesioned optic tract and act as prosthetic bridges to promote axonal regeneration. Nineteen rats (20-21 days old) were given hydrogel/Schwann cell implants. No obvious toxic effects were seen, either to the transplanted glia or in the adjacent host tissue. Schwann cells survived the implantation technique and were immunopositive for the low affinity nerve growth factor receptor, S100 and laminin. Immunohistochemical studies showed that host non-neuronal cells (astrocytes, oligodendroglia and macrophages) migrated into the implanted hydrogels. Astrocytes were the most frequently observed host cell in the polymer bridges. RT97-positive axons were seen in about two thirds of the implants. The axons were closely associated with transplanted Schwann cells and, in some cases, host glia (astrocytes). Individual axons regrowing within the implanted hydrogels could be traced for up to 900 microns, showing that there was continuity in the network of channels within the polymer scaffold. Axons did not appear to be myelinated by either Schwann cells or by migrated host oligodendroglia. In three rats, anterograde tracing with WGA/HRP failed to demonstrate the presence of retinal axons within the hydrogels. The data indicate that poly(HEMA) hydrogels containing Schwann cells have the potential to provide a stable three-dimensional scaffold which is capable of supporting axonal regeneration in the damaged CNS.

  View details for Web of Science ID A1995QG26100016

  View details for PubMedID 7728523

• REGROWTH OF AXONS WITHIN SCHWANN CELL-FILLED POLYCARBONATE TUBES IMPLANTED INTO THE DAMAGED OPTIC TRACT AND CEREBRAL-CORTEX OF RATS RESTORATIVE NEUROLOGY AND NEUROSCIENCE Harvey, A. R., Chen, M., Plant, G. W., Dyson, S. E. 1994; 6 (3): 221-237

  Abstract

  The efficacy of Schwann cell-filled polycarbonate tubes as a bridging substrate for regrowing axons following lesions of the rat optic tract or cerebral cortex has been assessed after short (11-31 days) or long (82-119 days) survival times. Tubes were impregnated with Iaminin and poly-L-lysine, soaked in basic FGF and filled with Schwann cells. They were implanted into optic tract lesions in 34 rats aged 15-21 days and into cortical lesion cavities in 3 adult rats. Gelfoam soaked in basic FGF and Schwann cell conditioned medium was placed over the tubes. In one group of rats, axon regrowth into implants was assessed using neurofilament antibody RT97; antibodies to proteolipid protein, Po, Iaminin, the low-affinity nerve growth factor receptor (NGFr), S-100 and EDI were also used to study myelination and the cellular content of the tubes. In a second group of rats, anterograde tracing techniques were used to specifically identify host retinal axons within the implanted polymers. After long survival times, the relationships between regrown axons and cells inside the tubes were also examined ultrastructurally. In all implants examined immunohistochemically at short survival times, large numbers of RT97+ axons were found throughout the tubes, usually in association with Iaminin+, NGFr+ Schwann cells. At longer survival times, viable Schwann cells were still present, but tubes contained fewer axons and less cellular material. This material often formed a cord (200-250 μm thick) which extended the length of the implant. In the second group of rats, labelled retinal axons were found in 11 of the 16 implants that were attached to the dLGN. Axons regrew up to 1 mm but did not reach the distal (tectal) end of the implants. Interestingly, there was no evidence of myelinogenesis by either implanted Schwann cells or by host-derived oligodendroglia which had migrated into the tubes. Oligodendroglia were usually encircled by processes, many of which originated from Schwann cells, suggesting that the grafted cells may have been involved in isolating the central glia. The data show that Schwann cell-filled polycarbonate tubes provided a favourable milieu for axonal regeneration in the short term; however over time there was a decrease in the cellular and fibre content of the tubes. After intracranial implantation, an additional supporting matrix inside the polycarbonate tubes may aid in providing an environment conducive to the long term maintenance of regenerated retinal and other axons.

  View details for Web of Science ID A1994NK44400006

  View details for PubMedID 21551752

• THE DISTRIBUTION OF ASTROCYTES, OLIGODENDROGLIA AND MYELIN IN NORMAL AND TRANSPLANTED RAT SUPERIOR COLLICULUS - AN IMMUNOHISTOCHEMICAL STUDY JOURNAL OF NEURAL TRANSPLANTATION & PLASTICITY Harvey, A. R., Plant, G. W., Kent, A. P. 1993; 4 (1): 1-14

  Abstract

  Immunohistochemical methods have been used to determine the distribution of macroglia and myelin in the normal rat superior colliculus (SC) and in grafts of fetal tectal tissue. The fetal tissue was derived from 15 day-old (E15) rat embryos and was transplanted onto the midbrain of newborn host rats of the same (PVG/c) strain. Antibodies to glial fibrillary acidic protein (GFAP) and carbonic anhydrase II (CAII) were used to visualize astrocytes and oligodendroglia respectively. Myelin was immunostained with antibodies to either proteolipid protein (PLP) or myelin basic protein (MBP). In the normal SC, GFAP positive astrocytes were found scattered throughout the SC, particularly in the superficial layers. They were especially prominent at the pial surface, around major blood vessels and at the midline between the two colliculi. CAII immunoreactive oligodendroglia and associated myelin were also found throughout the SC; by far the lowest density was seen in the stratum griseum superficiale (SGS). Both types of macroglia cell were found in abundance in tectal transplants, indicating that the precursors of these glial types were present in the E15 rat mesencephalon. In mature grafts, large numbers of fibrous astrocytes were found throughout the neuropil and the level of GFAP immunoreactivity was consistently greater than in host SC. Astrocytes seemed to be maintained in a reactive, perhaps immature state within the grafted tissue. Tectal transplants possessed large numbers of fully differentiated CAII-positive oligodendroglia and the grafts contained a dense network of myelinated axons. However the distribution of CAII and PLP immunoreactivity was not homogeneous; there were localized, well-defined regions that contained few oligodendroglia and relatively little myelin. These areas stained intensely for acetylcholinesterase (AChE) and were almost certainly homologous to the SGS of normal SC. The relative lack of oligodendroglia in the AChE stained patches in grafts and in SGS in situ suggests that local factors influencing the proliferation and distribution of oligodendroglia in normal SC may have been operating in a similar manner within the tectal transplant neuropil.

  View details for Web of Science ID A1993MG88700001

  View details for PubMedID 7509197