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Labeling of olfactory ensheathing glial cells with fluorescent tracers for neurotransplantation
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
2010 (English)In: Brain Research Bulletin, ISSN 0361-9230, E-ISSN 1873-2747, Vol. 81, no 1, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

Development of cell-based treatment strategies for repair of the injured nervous system requires cell tracing techniques to follow the fate of transplanted cells and their interaction with the host tissue. The present study investigates the efficacy of fluorescent cell tracers Fast Blue, PKH26, DiO and CMFDA for long-term labeling of olfactory ensheathing glial cells (OEC) in culture and following transplantation into the rat spinal cord. All tested dyes produced very efficient initial labeling of p75-positive OEC in culture. The number of Fast Blue-positive cells remained largely unchanged during the first 4 weeks but only about 21% of the cells retained tracer 6 weeks after labeling. In contrast, the number of cells labeled with PKH26 and DiO was reduced to 51-55% after 2 weeks in culture and reached 8-12% after 4-6 weeks. CMFDA had completely disappeared from the cells 2 weeks after labeling. AlamarBlue assay showed that among four tested tracers only CMFDA reduced proliferation rate of the OEC. After transplantation into spinal cord, Fast Blue-labeled OEC survived for at least 8 weeks but demonstrated very limited migration from the injection sites. Additional immunostaining with glial and neuronal markers revealed signs of dye leakage from the transplanted cells resulted in weak labeling of microglia and spinal neurons. The results show that Fast Blue is an efficient cell marker for cultured OEC. However, transfer of the dye from the transplanted cells to the host tissue should be considered and correctly interpreted.

Place, publisher, year, edition, pages
2010. Vol. 81, no 1, 125-132 p.
Keyword [en]
Olfactory ensheathing cells; Spinal cord; Transplantation; Fluorescent tracers; Long-term labeling
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:umu:diva-41359DOI: 10.1016/j.brainresbull.2009.10.005ISI: 000274101500020PubMedID: 19828127OAI: oai:DiVA.org:umu-41359DiVA: diva2:405680
Available from: 2011-03-23 Created: 2011-03-23 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Biosynthetic conduits and cell transplantation for neural repair
Open this publication in new window or tab >>Biosynthetic conduits and cell transplantation for neural repair
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Spinal cord injury results in complete failure of the central neurons to regenerate and is associated with cyst formation and enlargement of the trauma zone. In contrast to the spinal cord, axons in the injured peripheral nerve have the capacity to undergo some spontaneous regeneration. However, significant post-traumatic loss of nervous tissue causing long nerve gap is one of the main reasons for the poor restoration of function following microsurgical repair of injured nerves. The present thesis investigates the effects of biodegradable conduits prepared from fibrin glue and poly-beta-hydroxybutyrate (PHB) in combination with cultured Schwann cells, mesenchymal stem cells and extracellular matrix molecules on regeneration after spinal cord and peripheral nerve injury in adult rats.

At 4-8 weeks after transplantation into the injured spinal cord, the PHB conduit was well integrated into the cavity but regenerating axons were found mainly outside the PHB. When suspension of BrdU-labeled Schwann cells was added to the PHB, regenerating axons filled the conduit and became associated with the implanted cells. Modification of the PHB surface with extracellular matrix molecules significantly increased Schwann cell attachment and proliferation but did not alter axonal regeneration. To improve the labeling technique of the transplanted cells, the efficacy of fluorescent cell tracers Fast Blue, PKH26, Vibrant DiO and Cell Tracker™ Green CMFDA was evaluated. All tested dyes produced very efficient initial labeling of olfactory ensheathing glial cells in culture. The number of Fast Blue-labeled cells remained largely unchanged during the first 4 weeks whereas the number of cells labeled with other tracers was significantly reduced after 2 weeks. After transplantation into the spinal cord, Fast Blue-labeled glial cells survived for 8 weeks but demonstrated very limited migration from the injection sites. Additional immunostaining with glial and neuronal markers demonstrated transfer of the dye from the transplanted cells to the host tissue.

In a sciatic nerve injury model, the extent of axonal regeneration through a 10mm gap bridged with tubular PHB conduit was compared with a fibrin glue conduit. At 2 weeks after injury, the fibrin conduit supported similar axonal regeneration and migration of the host Schwann cells compared with the PHB conduit augmented with a diluted fibrin matrix and GFP-labeled Schwann cells or mesenchymal stem cells. The long-term regenerative response was evaluated using retrograde neuronal labeling. The fibrin glue conduit promoted regeneration of 60% of sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed more chaotic appearance and the mean area and diameter of fast type fibers were significantly worse than those of the corresponding 10mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size.

In summary, these results show that a PHB conduit promotes attachment, proliferation and survival of adult Schwann cells and supports marked axonal growth after transplantation into the injured spinal cord. The data suggest an advantage of the fibrin conduit for the important initial phase of peripheral nerve regeneration and demonstrate potential of the conduit to promote long-term neuronal regeneration and muscle recovery.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2011. 61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1408
Keyword
Spinal cord injury; Peripheral nerve injury; Nerve graft; Biosynthetic conduit; Nerve tissue engineering; Neural prosthesis
National Category
Neurosciences
Research subject
Neurology; Neurosurgery
Identifiers
urn:nbn:se:umu:diva-42440 (URN)978-91-7459-160-6 (ISBN)
Public defence
2011-05-19, BiA201, Biologihuset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2011-04-28 Created: 2011-04-07 Last updated: 2011-04-28Bibliographically approved

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