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Neuroprotection in the Injured Spinal Cord: Novel Strategies using Immunomodulation, Stem cell Transplantation and Hyaluronic acid Hydrogel carriers
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics. (The SpineLab)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The overall aim of this thesis was to establish strategies to minimize secondary damage to the injured spinal cord. Secondary damage that follows spinal cord injury (SCI) involves inflammatory and excitotoxic pathways. Regulation of these pathways using immunomodulatory and neuroprotective substances potentially protects the injured spinal cord from further damage. We also developed and studied resorbable biomaterials to be used as carriers for potential neuroprotectants to the injured spinal cord.

We used transversal spinal cord slice cultures (SCSCs) derived from postnatal mice as a model. SCSCs were maintained on different biomaterials and were studied after treatment with immunomodulatory and/or neurotrophic factors. They were further excitotoxically injured and subsequently treated with interleukin-1 receptor antagonist (IL1RA) or by neural crest stem cell (NCSC)-transplantation.

The results show that biocompatible and resorbable hydrogels based on hyaluronic acid (HA) preserved neurons in SCSCs to a much higher extent than a conventional collagen-based biomaterial or standard polyethylene terephthalate (PET) membrane inserts. Glial activation was limited in the cultures maintained on HA-based hydrogel. The anti-inflammatory factor IL1RA protected SCSCs from degenerative mechanisms that occur during in vitro incubation, and IL1RA also protected SCSCs from excitotoxic injury induced by N-Methyl-d-Aspartate (NMDA). IL1RA specifically protected neurons that resided in the ventral horn, while other neuronal populations such as dorsal horn neurons and Renshaw cells did not respond to treatment. Finally, transplantation of NCSCs onto excitotoxically injured SCSCs protected from neuronal loss, apoptosis and glial activation, while NCSCs remained undifferentiated.

The results presented in this thesis indicate that carriers based on HA seem to be more suitable than conventional collagen-based biomaterials since they enhance neuronal survival per se. The observed neuroprotection is likely due to biomechanical properties of HA. IL1RA protects SCSCs from spontaneous degeneration and from NMDA-induced injury, suggesting that excitotoxic mechanisms can be modulated through anti-inflammatory pathways. Different neuronal populations are affected by IL1RA to various degrees, suggesting that a combination of different neuroprotectants should be used in treatment strategies after SCI. Finally, NCSCs seem to protect SCSCs from excitotoxic injury through paracrine actions, since they remain undifferentiated and do not migrate into the tissue during in vitro incubation.

It seems that combinations of neuroprotectants and carrier substances should be considered rather than one single strategy when designing future treatments for SCI. Incorporation of neuroprotectants such as IL1RA combined with stem cells in injectable biocompatible carriers based on HA is the final goal of our group in the treatment of SCI.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1106
Keyword [en]
Hyaluronic Acid-based hydrogel, motorneurons, microglial cells, Interleukin-1 Receptor Antagonist, Renshaw cells, excitotoxicity, neuroinflammation, Neural Crest Stem Cells
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-251477ISBN: 978-91-554-9255-7 (print)OAI: oai:DiVA.org:uu-251477DiVA: diva2:806232
Public defence
2015-06-12, Grönwallsalen, Akademiska sjukhuset, Akademiska sjukhuset ing.70, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-06-04 Created: 2015-04-19 Last updated: 2016-04-21
List of papers
1. Hyaluronic acid-based hydrogel enhances neuronal survival in spinal cord slice cultures from postnatal mice
Open this publication in new window or tab >>Hyaluronic acid-based hydrogel enhances neuronal survival in spinal cord slice cultures from postnatal mice
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2014 (English)In: Journal of biomaterials applications, ISSN 0885-3282, E-ISSN 1530-8022, Vol. 28, no 6, 825-836 p.Article in journal (Refereed) Published
Abstract [en]

Numerous biomaterials based on extracellular matrix-components have been developed. It was our aim to investigate whether a hyaluronic acid-based hydrogel improves neuronal survival and tissue preservation in organotypic spinal cord slice cultures. Organotypic spinal cord slice cultures were cultured for 4 days in vitro (div), either on hyaluronic acid-based hydrogel (hyaluronic acid-gel group), collagen gel (collagen group), directly on polyethylene terephthalate membrane inserts (control group), or in the presence of soluble hyaluronic acid (soluble hyaluronic acid group). Cultures were immunohistochemically stained against neuronal antigen NeuN and analyzed by confocal laser scanning microscopy. Histochemistry for choline acetyltransferance, glial fibrillary acidic protein, and Griffonia simplicifolia isolectin B4 followed by quantitative analysis was performed to assess motorneurons and different glial populations. Confocal microscopic analysis showed a 4-fold increase in the number of NeuN-positive neurons in the hyaluronic acid-gel group compared to both collagen (p < 0.001) and control groups (p < 0.001). Compared to controls, organotypic spinal cord slice cultures maintained on hyaluronic acid-based hydrogel showed 5.9-fold increased survival of choline acetyltransferance-positive motorneurons (p = 0.008), 2-fold more numerous resting microglial cells in the white matter (p = 0.031), and a 61.4% reduction in the number of activated microglial cells within the grey matter (p = 0.05). Hyaluronic acid-based hydrogel had a shear modulus (G') of ≈1200 Pascals (Pa), which was considerably higher than the ≈25 Pa measured for collagen gel. Soluble hyaluronic acid failed to improve tissue preservation. In conclusion, hyaluronic acid-based hydrogel improves neuronal and - most notably - motorneuron survival in organotypic spinal cord slice cultures and microglial activation is limited. The positive effects of hyaluronic acid-based hydrogel may at least in part be due to its mechanical properties.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-216218 (URN)10.1177/0885328213483636 (DOI)000329317100003 ()23674184 (PubMedID)
Available from: 2014-01-20 Created: 2014-01-20 Last updated: 2017-12-06Bibliographically approved
2. Interleukin-1 Receptor Antagonist Promotes Survival of Ventral Horn Neurons and Suppresses Microglial Activation in Mouse Spinal Cord Slice Cultures
Open this publication in new window or tab >>Interleukin-1 Receptor Antagonist Promotes Survival of Ventral Horn Neurons and Suppresses Microglial Activation in Mouse Spinal Cord Slice Cultures
2014 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 92, no 11, 1457-1465 p.Article in journal (Refereed) Published
Abstract [en]

Secondary damage after spinal cord injury (SCI) induces neuronal demise through neurotoxicity and inflammation, and interleukin (IL)-1 beta is a key inflammatory mediator. We hypothesized that IL-1 beta is released in spinal cord slice cultures (SCSC) and aimed at preventing the potentially neurotoxic effects of IL-1 beta by using interleukin-1 receptor antagonist (IL1RA). We hypothesized that IL1RA treatment enhances neuronal survival and suppresses microglial activation. SCSC were cultured up to 8 days in vitro (DIV) in the presence of IL1RA or without, either combined with trophic support using neurotrophin (NT)-3 or not. Four groups were studied: negative control, IL1RA, NT-3, and IL1RA1NT-3. IL-1 beta concentrations in supernatants were measured by ELISA. SCSC were immunohistochemically stained for NeuN and a-neurofilament, and microglial cells were visualized with isolectin B-4. After 8 DIV, ventral horn neurons were significantly more numerous in the IL1RA, NT-3, and IL1RA1NT-3 groups compared with negative controls. Activated microglial cells were significantly less numerous in the IL1RA, NT-3, and IL1RA1NT-3 groups compared with negative controls. Axons expanded into the collagen matrix after treatment with IL1RA, NT-3, or IL1RA1NT-3, but not in negative controls. IL-1 beta release from cultures peaked after 6 hr and was lowest in the IL1RA1NT-3 group. We conclude that IL-1 beta is released in traumatized spinal cord tissue and that IL1RA could exert its neuroprotective actions by blocking IL-1-receptors. IL1RA thereby sustains neuronal survival irrespective of the presence of additional trophic support. Microglial activation is suppressed in the presence of IL1RA, suggesting decreased inflammatory activity. IL1RA treatment approaches may have substantial impact following SCI.

Keyword
interleukin-1, neuroinflammation, neuroprotection
National Category
Neurology Neurosciences
Identifiers
urn:nbn:se:uu:diva-231972 (URN)10.1002/jnr.23429 (DOI)000340534900005 ()
Available from: 2014-09-15 Created: 2014-09-12 Last updated: 2017-12-05Bibliographically approved
3. Differential Neuroprotective Effects of Interleukin-1 Receptor Antagonist on Spinal Cord Neurons after Excitotoxic Injury
Open this publication in new window or tab >>Differential Neuroprotective Effects of Interleukin-1 Receptor Antagonist on Spinal Cord Neurons after Excitotoxic Injury
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(English)Manuscript (preprint) (Other academic)
Keyword
Excitotoxicity, neuroprotection, motor neurons, Chrna2, microglial cells
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-251475 (URN)
Note

Secondary damage following spinal cord injury (SCI) induces neuronal damage through inflammatory and excitotoxic pathways. We hypothesized that the interleukin-1 receptor antagonist (IL1RA) can protect ventral horn neurons after excitotoxic neuronal damage in spinal cord slice cultures (SCSC). These cultures were subjected to excitotoxic injury with N-Methyl-D-Aspartate (NMDA) and treated with IL1RA. Immunohistochemistry for neuronal nuclei (NeuN), MacII, glialfibrillary- acidic-protein (GFAP), and TUNEL stains were used to evaluate neuronal survival, glial activation and apoptosis. Treatment with IL1RA dramatically reduced the number of apoptotic cells in both NMDA-lesioned and unlesioned cultures. More specifically, NMDA reduced the number of NeuN-positive ventral horn neurons and IL1RA treatment restored their number one day post injury. However, IL1RA had no effect on the number of presumable Renshaw cells, based on their selective expression of the cholinergic nicotinic alpha-2 receptor subunit (Chrna2). Moreover, activated microglial cells were more numerous in NMDA-lesioned cultures one day after injury and IL1RA significantly reduced their number. We conclude that IL1RA inhibits neuronal apoptosis and microglial activation in the excitotoxically injured spinal cord. Renshaw cells were more susceptible to excitotoxic injury than other neurons and were not rescued by IL1RA treatment. Modulation of IL-1-mediated pathways may thus be effective in reducing excitotoxically induced neuronal damage after SCI, although its neuroprotective effects seem to be differential

Available from: 2015-04-19 Created: 2015-04-19 Last updated: 2015-07-07
4. Neural Crest Stem Cells protect Spinal Cord Slice Cultures from Excitotoxic Neuronal Damage and Inhibit Glial Activation
Open this publication in new window or tab >>Neural Crest Stem Cells protect Spinal Cord Slice Cultures from Excitotoxic Neuronal Damage and Inhibit Glial Activation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Neural Crest Stem Cells (NCSC) possess anti-inflammatory properties and they could therefore have protective effects on neurons after spinal cord injury (SCI). We investigated if NCSCs reduce neuronal loss and glial activation after excitotoxic injury in spinal cord slice cultures (SCSCs). SCSCs subjected to N-Methyl-D-Aspartate (NMDA)-induced injury were either treated by transplantation of NCSC or with interleukin-1 receptor antagonist (IL1RA). Immunohistochemistry for Neuronal Nuclei (NeuN), glial fibrillary acidic protein (GFAP) and MacII were used to study neuronal and glial populations, and TUNEL staining to visualize apoptotic cells. Migration and differentiation of NCSCs on SCSCs, laminin or a hyaluronic acid hydrogel were investigated by immunohistochemistry. NCSCs counteracted the neuronal loss observed after NMDA-induced excitotoxicity and reduced the proportion of apoptotic cells. Transplantation of NCSCs also reduced the number of activated microglial cells in the white matter and the number of activated astrocytes in the grey matter of NMDA-injured SCSCs. The majority of NCSCs migrated superficially across the surface of SCSCs with some of them penetrating into the tissue. NCSCs did not show signs of neuronal or glial differentiation, however, the markers SOX2 and Krox20 were expressed which indicated an undifferentiated state of NCSCs. In conclusion, NCSCs have neuroprotective, anti-apoptotic and anti-inflammatory effects on SCSCs subjected to excitotoxicity, similar to the effects of IL1RA. Since the majority of NCSCs did not migrate through SCSCs they are likely to exert their actions through the secretion of soluble factors.

Keyword
Neuroprotection, Suppressed glial activation, excitotoxicity, apoptosis
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-251476 (URN)
Available from: 2015-04-19 Created: 2015-04-19 Last updated: 2015-07-07

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