Recent advances in the understanding of the aetiology and therapeutic strategies in burning mouth syndrome: Focus on the actions of cannabinoids.

Burning mouth syndrome (BMS) is a neuropathic pain disorder associated with a burning sensation on oral mucosal surfaces with frequently reported xerostomia, dysgeusia and tingling or paraesthetic sensations. However, patients present no clinically evident causative lesions. The poor classification of the disorder has resulted in a diagnostic challenge, particularly for the clinician/dentist evaluating these individuals. Major research developments have been made in the BMS field in recent years to address this concern, principally in terms of the pathophysiological mechanisms underlying the disorder, in addition to therapeutic advancements. For the purpose of this review, an update on the pathophysiological mechanisms will be discussed from a neuropathic, immunological, hormonal and psychological perspective. This review will also focus on the many therapeutic strategies that have been explored for BMS, including antidepressants/antipsychotics, non-steroidal anti-inflammatories, hormone replacement therapies, phytotherapeutic compounds and non-pharmacological interventions, overall highlighting the lack of controlled clinical studies to support the effectiveness of such therapeutic avenues. Particular focus is given to the cannabinoid system and the potential of cannabis-based therapeutics in managing BMS patients.

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

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.

Three-dimensional cell culture can be regulated by vibration: low-frequency vibration increases the size of olfactory ensheathing cell spheroids.

BACKGROUND: Olfactory ensheathing cell (OEC) transplantation is emerging as a promising therapy for spinal cord injuries. However, outcomes are inconsistent, and the method needs improvement. Currently, cells are injected into the injury site as a suspension, and often fail to form a three-dimensional (3D) network crucial for both survival of the transplanted cells, and for regeneration of severed axons. 3D culture systems are therefore likely to improve the method. Of the many 3D culture systems available, the spheroid-producing naked liquid marble (NLM) technique is particularly advantageous compared to other platforms as it rapidly generates cell spheroids which can easily be extracted for further handling. To improve production of the spheroids, we designed and tested a device which allows fine control over vibrational stimuli to liquid marble cell cultures. We applied vibrational frequencies of 20, 60, and 80 Hz with consistent amplitude to NLM containing OECs and assessed the size and number of the 3D cell spheroids generated as well as the migratory capacity of cells cultured in the vibrated spheroids. RESULTS: Vibrating the NLMs led to fewer and dramatically larger spheroids in comparison to non-vibrated NLMs. Of the frequencies tested, 60 Hz caused over 70-fold increase in spheroid volume. When transferred to a culture plate, the larger spheroids retained their structure after 72 h in culture, and cells that migrated out of the spheroids covered a significantly larger area compared to cells migrating out of spheroids formed at all the other frequencies tested. CONCLUSIONS: We have shown that vibration can be used to regulate the formation of cell spheroids in NLM cultures. The ability to modulate the size of spheroids is useful for a range of 3D cell culture models and for preparing cells for in vivo transplantation.

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

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

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."

The serrulatane diterpenoid natural products RAD288 and RAD289 stimulate properties of olfactory ensheathing cells useful for neural repair therapies.

Olfactory ensheathing cells (OECs) are being trialled for cell transplantation therapies for neural repair as they have unique properties which can enhance neuron regeneration. However, improvements in cell viability, proliferation and migration are needed to enhance therapeutic outcomes. Growth factors can enhance cell activity, but they can also induce side effects as they can act on numerous cell types. An alternative approach is to identify natural products (NPs) that more selectively activate specific cell functions. We have examined two pure NPs, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid (RAD288) and 3,7,8-trihydroxyserrulat-14-en-19-oic acid (RAD289) isolated from the Australian plant Eremophila microtheca. We determined that RAD288 and RAD289 stimulated the viability and proliferation of OECs in two-dimensional cultures and increased cell viability in three-dimensional spheroids. Both compounds also enhanced OEC-mediated phagocytosis of neural debris. However, only RAD288 stimulated migration of OECs, demonstrating that key structural changes to the compound can dramatically affect the resultant cellular action. In addition, cell-type specific action is highlighted by the result that neither compound stimulated the viability of Schwann cells which are a closely-related glial cell type. Therefore, these small molecules may have high potential for selective activation of specific therapeutically-useful activities of OECs for transplantation therapies to repair the nervous system.

Linckosides enhance proliferation and induce morphological changes in human olfactory ensheathing cells.

Linckosides are members of the steroid glycoside family isolated from the starfish Linckia laevigata. These natural compounds have notable neuritogenic activity and synergistic effects on NGF-induced neuronal differentiation of PC12 cells. Neurogenic factors or molecules that are able to mimic their activities are known to be involved in the survival, proliferation and migration of neurons and glial cells; however how glial cells respond to specific neurogenic molecules such as linckosides has not been investigated. This study aimed to examine the effect of three different linckosides (linckoside A, B and granulatoside A) on the morphological properties, proliferation and migration of human olfactory ensheathing cells (hOECs). The proliferation rate after all the treatments was higher than control as detected by MTS assay. Additionally, hOECs displayed dramatic morphological changes characterized by a higher number of processes after linckoside treatment. Interestingly changes in microtubule organization and expression levels of some early neuronal markers (GAP43 and ßIII-tubulin) were also observed. An increase in the phosphorylation of ERK 1/2 after addition of the compounds suggests that this pathway may be involved in the linckoside-mediated effects particularly those related to morphological changes. These results are the first description of the stimulating effects of linckosides on hOECs and raise the potential for this natural compound or its derivatives to be used to regulate and enhance the therapeutic properties of OECs, particularly for cell transplantation therapies.

Mechanism of impaired microtubule-dependent peroxisome trafficking and oxidative stress in SPAST-mutated cells from patients with Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is an inherited neurological condition that leads to progressive spasticity and gait abnormalities. Adult-onset HSP is most commonly caused by mutations in SPAST, which encodes spastin a microtubule severing protein. In olfactory stem cell lines derived from patients carrying different SPAST mutations, we investigated microtubule-dependent peroxisome movement with time-lapse imaging and automated image analysis. The average speed of peroxisomes in patient-cells was slower, with fewer fast moving peroxisomes than in cells from healthy controls. This was not because of impairment of peroxisome-microtubule interactions because the time-dependent saltatory dynamics of movement of individual peroxisomes was unaffected in patient-cells. Our observations indicate that average peroxisome speeds are less in patient-cells because of the lower probability of individual peroxisome interactions with the reduced numbers of stable microtubules: peroxisome speeds in patient cells are restored by epothilone D, a tubulin-binding drug that increases the number of stable microtubules to control levels. Patient-cells were under increased oxidative stress and were more sensitive than control-cells to hydrogen peroxide, which is primarily metabolised by peroxisomal catalase. Epothilone D also ameliorated patient-cell sensitivity to hydrogen-peroxide. Our findings suggest a mechanism for neurodegeneration whereby SPAST mutations indirectly lead to impaired peroxisome transport and oxidative stress.

Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles.

We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

Differing phagocytic capacities of accessory and main olfactory ensheathing cells and the implication for olfactory glia transplantation therapies.

The rodent olfactory systems comprise the main olfactory system for the detection of odours and the accessory olfactory system which detects pheromones. In both systems, olfactory axon fascicles are ensheathed by olfactory glia, termed olfactory ensheathing cells (OECs), which are crucial for the growth and maintenance of the olfactory nerve. The growth-promoting and phagocytic characteristics of OECs make them potential candidates for neural repair therapies such as transplantation to repair the injured spinal cord. However, transplanting mixed populations of glia with unknown properties may lead to variations in outcomes for neural repair. As the phagocytic capacity of the accessory OECs has not yet been determined, we compared the phagocytic capacity of accessory and main OECs in vivo and in vitro. In normal healthy animals, the accessory OECs accumulated considerably less axon debris than main OECs in vivo. Analysis of freshly dissected OECs showed that accessory OECs contained 20% less fluorescent axon debris than main OECs. However, when assayed in vitro with exogenous axon debris added to the culture, the accessory OECs phagocytosed almost 20% more debris than main OECs. After surgical removal of one olfactory bulb which induced the degradation of main and accessory olfactory sensory axons, the accessory OECs responded by phagocytosing the axon debris. We conclude that while accessory OECs have the capacity to phagocytose axon debris, there are distinct differences in their phagocytic capacity compared to main OECs. These distinct differences may be of importance when preparing OECs for neural transplant repair therapies.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system.

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100ß-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

Low-dose curcumin stimulates proliferation, migration and phagocytic activity of olfactory ensheathing cells.

One of the promising strategies for neural repair therapies is the transplantation of olfactory ensheathing cells (OECs) which are the glial cells of the olfactory system. We evaluated the effects of curcumin on the behaviour of mouse OECs to determine if it could be of use to further enhance the therapeutic potential of OECs. Curcumin, a natural polyphenol compound found in the spice turmeric, is known for its anti-cancer properties at doses over 10 µM, and often at 50 µM, and it exerts its effects on cancer cells in part by activation of MAP kinases. In contrast, we found that low-dose curcumin (0.5 µM) applied to OECs strikingly modulated the dynamic morphology, increased the rate of migration by up to 4-fold, and promoted significant proliferation of the OECs. Most dramatically, low-dose curcumin stimulated a 10-fold increase in the phagocytic activity of OECs. All of these potently stimulated behavioural characteristics of OECs are favourable for neural repair therapies. Importantly, low-dose curcumin gave a transient activation of p38 kinases, which is in contrast to the high dose curcumin effects on cancer cells in which these MAP kinases tend to undergo prolonged activation. Low-dose curcumin mediated effects on OECs demonstrate cell-type specific stimulation of p38 and ERK kinases. These results constitute the first evidence that low-dose curcumin can modulate the behaviour of olfactory glia into a phenotype potentially more favourable for neural repair and thereby improve the therapeutic use of OECs for neural repair therapies.