Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Griffith University

Olfactory ensheathing cells (OECs) are often described as being present in both the peripheral and the central nervous systems (PNS and CNS). Furthermore, the olfactory nervous system glia limitans (the glial layer defining the PNS-CNS border) is considered unique as it consists of intermingling OECs and astrocytes. In contrast, the glia limitans of the rest of the nervous system consists solely of astrocytes which create a distinct barrier to Schwann cells (peripheral glia). The ability of OECs to interact with astrocytes is one reason why OECs are believed to be superior to Schwann cells for transplantation therapies to treat CNS injuries. We have used transgenic reporter mice in which glial cells express DsRed fluorescent protein to study the cellular constituents of the glia limitans. We found that the glia limitans layer of the olfactory nervous system is morphologically similar to elsewhere in the nervous system, with a similar low degree of intermingling between peripheral glia and astrocytes. We found that the astrocytic layer of the olfactory bulb is a distinct barrier to bacterial infection, suggesting that this layer constitutes the PNS-CNS immunological barrier. We also found that OECs interact with astrocytes in a similar fashion as Schwann cells in vitro. When cultured in three dimensions, however, there were subtle differences between OECs and Schwann cells in their interactions with astrocytes. We therefore suggest that glial fibrillary acidic protein-reactive astrocyte layer of the olfactory bulb constitutes the glia limitans of the olfactory nervous system and that OECs are primarily "PNS glia."

Novel insights into the glia limitans of the olfactory nervous system.

Three-dimensional (3D) multicellular structures allow cells to behave and interact with each other in a manner that mimics the in vivo environment. In recent years, many 3D cell culture methods have been developed with the goal of producing the most in vivo-like structures possible. Whilst strongly preferable to  conventional cell culture, these approaches are often poorly reproducible, time-consuming, expensive, and labor-intensive and require specialized equipment. Here, we describe a novel 3D culture platform, which we have termed the naked liquid marble (NLM). Cells are cultured in a liquid drop (the NLM) in superhydrophobic-coated plates, which causes the cells to naturally form 3D structures. Inside the NLMs, cells are free to interact with each other, forming multiple 3D spheroids that are uniform in size and shape in less than 24 h. We showed that this system is highly reproducible, suitable for cell coculture, compound screening, and also compatible with laboratory automation systems. The low cost of production, small volume of each NLM, and production via automated liquid handling make this 3D cell-culturing system particularly suitable for high-throughput screening assays such as drug testing as well as numerous other cell-based research applications.

Naked Liquid Marbles: A Robust Three-Dimensional Low-Volume Cell-Culturing System.

Olfactory ensheathing cells (OECs), the glial cells of the primary olfactory nervous system, support the natural regeneration of the olfactory nerve that occurs throughout life. OECs thus exhibit unique properties supporting neuronal survival and growth. Transplantation of OECs is emerging as a promising treatment for spinal cord injury; however, outcomes in both animals and humans are variable and the method needs improvement and standardization. A major reason for the discrepancy in functional outcomes is the variability in survival and integration of the transplanted cells, key factors for successful spinal cord regeneration. Here, we review the outcomes of OEC transplantation in rodent models over the last 10 years, with a focus on survival and integration of the transplanted cells. We identify the key factors influencing OEC survival: injury type, source of transplanted cells, co-transplantation with other cell types, number and concentration of cells, method of delivery, and time of transplantation after the injury. We found that two key issues are hampering optimization and standardization of OEC transplantation: lack of (1) reliable methods for identifying transplanted cells, and (2) three-dimensional systems for OEC delivery. To develop OEC transplantation as a successful and standardized therapy for spinal cord injury, we must address these issues and increase our understanding of the complex parameters influencing OEC survival.

Survival and Integration of Transplanted Olfactory Ensheathing Cells are Crucial for Spinal Cord Injury Repair: Insights from the Last 10 Years of Animal Model Studies.

The infectious disease melioidosis is caused by the bacterium Burkholderia pseudomallei. Melioidosis is characterised by high mortality and morbidity and can involve the central nervous system (CNS). We have previously discovered that B. pseudomallei can infect the CNS via the olfactory and trigeminal nerves in mice. We have shown that the nerve path is dependent on mouse strain, with outbred mice showing resistance to olfactory nerve infection. Damage to the nasal epithelium by environmental factors is common, and we hypothesised that injury to the olfactory epithelium may increase the vulnerability of the olfactory nerve to microbial insult. We therefore investigated this, using outbred mice that were intranasally inoculated with B. pseudomallei, with or without methimazole-induced injury to the olfactory neuroepithelium. Methimazole-mediated injury resulted in increased B. pseudomallei invasion of the olfactory epithelium, and only in pre-injured animals were bacteria found in the olfactory nerve and bulb. In vitro assays demonstrated that B. pseudomallei readily infected glial cells isolated from the olfactory and trigeminal nerves (olfactory ensheathing cells and trigeminal Schwann cells, respectively). Bacteria were degraded by some cells but persisted in other cells, which led to the formation of multinucleated giant cells (MNGCs), with olfactory ensheathing cells less likely to form MNGCs than Schwann cells. Double Cap mutant bacteria, lacking the protein BimA, did not form MNGCs. These data suggest that injuries to the olfactory epithelium expose the primary olfactory nervous system to bacterial invasion, which can then result in CNS infection with potential pathogenic consequences for the glial cells.

Burkholderia pseudomallei invades the olfactory nerve and bulb after epithelial injury in mice and causes the formation of multinucleated giant glial cells in vitro.

Megha Shah

Menzies Health Institute Queensland,
Clem Jones Centre for Neurobiology and Stem Cell Research

Griffith University

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Megha Shah

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Griffith University

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Menzies Health Institute Queensland,
Clem Jones Centre for Neurobiology and Stem Cell Research