Liraglutide modulates adhesion molecules and enhances cell properties in three-dimensional cultures of olfactory ensheathing cells.

Cell transplantation using olfactory ensheathing cells (OECs) is a promising approach for nerve repair but there are numerous limitations with their delivery method. Three-dimensional (3D) cell culture systems potentially offer a powerful approach for cell production and delivery options. To further optimise the use of OECs, strategies to promote cell viability and maintain cell behaviours in 3D cultures become important. We previously demonstrated an anti-diabetic drug, liraglutide, could modulate OEC migration and re-model extracellular matrix in two-dimensional (2D) cultures. In the present study, we further investigated its beneficial effects in our 3D culture system using primary OECs. OECs treated with liraglutide at 100 nM showed improved cell viability and had modulated expression of N-cadherin and ß1-integrin (two important cell adhesion molecules). When formed into 3D spheroids, the pre-treated OECs generated spheroids with an increased volume and a decreased cell density compared to control spheroids. OECs that subsequently migrated out of the liraglutide pre-treated spheroids had higher capacity for migration with increased duration and length, which was attributed to a reduction in the pauses during the migration. Moreover, OECs that migrated out from liraglutide spheroids had a more bipolar morphology consistent with higher migratory capacity. In summary, liraglutide improved the viability of OECs, modulated cell adhesion molecules, and resulted in stable 3D cell constructs which conferred enhanced migratory capacity on the OECs. Overall, liraglutide may potentially improve the therapeutic use of OECs for neural repair by enhancing the generation of stable 3D constructs and increasing the migratory behaviour of OECs.

Designing a Clinical Trial with Olfactory Ensheathing Cell Transplantation-Based Therapy for Spinal Cord Injury: A Position Paper.

Spinal cord injury (SCI) represents an urgent unmet need for clinical reparative therapy due to its largely irreversible and devastating effects on patients, and the tremendous socioeconomic burden to the community. While different approaches are being explored, therapy to restore the lost function remains unavailable. Olfactory ensheathing cell (OEC) transplantation is a promising approach in terms of feasibility, safety, and limited efficacy; however, high variability in reported clinical outcomes prevent its translation despite several clinical trials. The aims of this position paper are to present an in-depth analysis of previous OEC transplantation-based clinical trials, identify existing challenges and gaps, and finally propose strategies to improve standardization of OEC therapies. We have reviewed the study design and protocols of clinical trials using OEC transplantation for SCI repair to investigate how and why the outcomes show variability. With this knowledge and our experience as a team of biologists and clinicians with active experience in the field of OEC research, we provide recommendations regarding cell source, cell purity and characterisation, transplantation dosage and format, and rehabilitation. Ultimately, this position paper is intended to serve as a roadmap to design an effective clinical trial with OEC transplantation-based therapy for SCI repair.

Designing Olfactory Ensheathing Cell Transplantation Therapies: Influence of Cell Microenvironment.

Olfactory ensheathing cell (OEC) transplantation is emerging as a promising treatment option for injuries of the nervous system. OECs can be obtained relatively easily from nasal biopsies, and exhibit several properties such as secretion of trophic factors, and phagocytosis of debris that facilitate neural regeneration and repair. But a major limitation of OEC-based cell therapies is the poor survival of transplanted cells which subsequently limit their therapeutic efficacy. There is an unmet need for approaches that enable the in vitro production of OECs in a state that will optimize their survival and integration after transplantation into the hostile injury site. Here, we present an overview of the strategies to modulate OECs focusing on oxygen levels, stimulating migratory, phagocytic, and secretory properties, and on bioengineering a suitable environment in vitro.

Macrophages Treated with VEGF and PDGF Exert Paracrine Effects on Olfactory Ensheathing Cell Function.

Glial cell transplantation using olfactory ensheathing cells (OECs) holds a promising approach for treating spinal cord injury (SCI). However, integration of OECs into the hostile acute secondary injury site requires interaction and response to macrophages. Immunomodulation of macrophages to reduce their impact on OECs may improve the functionality of OECs. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), known for their immunomodulatory and neuroprotective functions, have provided improved outcomes in SCI animal models. Thus, VEGF and PDGF modulation of the SCI microenvironment may be beneficial for OEC transplantation. In this in vitro study, the effect of VEGF and PDGF on macrophages in an inflammatory condition was tested. Combined VEGF + PDGF reduced translocation nuclear factor kappa B p65 in macrophages without altering pro-inflammatory cytokines. Further, the ability of OECs to phagocytose myelin debris was assessed using macrophage-conditioned medium. Conditioned medium from macrophages incubated with PDGF and combined VEGF + PDGF in inflammatory conditions promoted phagocytosis by OECs. The growth factor treated conditioned media also modulated the expression of genes associated with nerve repair and myelin expression in OECs. Overall, these results suggest that the use of growth factors together with OEC transplantation may be beneficial in SCI therapy.

Streptococcus agalactiae Infects Glial Cells and Invades the Central Nervous System via the Olfactory and Trigeminal Nerves.

Streptococcus agalactiae causes neonatal meningitis and can also infect the adult central nervous system (CNS). S. agalactiae can cross the blood-brain barrier but may also reach the CNS via other paths. Several species of bacteria can directly invade the CNS via the olfactory and trigeminal nerves, which extend between the nasal cavity and brain and injury to the nasal epithelium can increase the risk/severity of infection. Preterm birth is associated with increased risk of S. agalactiae infection and with nasogastric tube feeding. The tubes, also used in adults, can cause nasal injuries and may be contaminated with bacteria, including S. agalactiae. We here investigated whether S. agalactiae could invade the CNS after intranasal inoculation in mice. S. agalactiae rapidly infected the olfactory nerve and brain. Methimazole-mediated model of nasal epithelial injury led to increased bacterial load in these tissues, as well as trigeminal nerve infection. S. agalactiae infected and survived intracellularly in cultured olfactory/trigeminal nerve- and brain-derived glia, resulting in cytokine production, with some differences between glial types. Furthermore, a non-capsulated S. agalactiae was used to understand the role of capsule on glial cells interaction. Interestingly, we found that the S. agalactiae capsule significantly altered cytokine and chemokine responses and affected intracellular survival in trigeminal glia. In summary, this study shows that S. agalactiae can infect the CNS via the nose-to-brain path with increased load after epithelial injury, and that the bacteria can survive in glia.

Neuron-fibrous scaffold interfaces in the peripheral nervous system: a perspective on the structural requirements.

The nerves of the peripheral nervous system are not able to effectively regenerate in cases of severe neural injury. This can result in debilitating consequences, including morbidity and lifelong impairments affecting the quality of the patient's life. Recent findings in neural tissue engineering have opened promising avenues to apply fibrous tissue-engineered scaffolds to promote tissue regeneration and functional recovery. These scaffolds, known as neural scaffolds, are able to improve neural regeneration by playing two major roles, namely, by being a carrier for transplanted peripheral nervous system cells or biological cues and by providing structural support to direct growing nerve fibers towards the target area. However, successful implementation of scaffold-based therapeutic approaches calls for an appropriate design of the neural scaffold structure that is capable of up- and down-regulation of neuron-scaffold interactions in the extracellular matrix environment. This review discusses the main challenges that need to be addressed to develop and apply fibrous tissue-engineered scaffolds in clinical practice. It describes some promising solutions that, so far, have shown to promote neural cell adhesion and growth and a potential to repair peripheral nervous system injuries.

Advances in Electrospun Nerve Guidance Conduits for Engineering Neural Regeneration.

Injuries to the peripheral nervous system result in devastating consequences with loss of motor and sensory function and lifelong impairments. Current treatments have largely relied on surgical procedures, including nerve autografts to repair damaged nerves. Despite improvements to the surgical procedures over the years, the clinical success of nerve autografts is limited by fundamental issues, such as low functionality and mismatching between the damaged and donor nerves. While peripheral nerves can regenerate to some extent, the resultant outcomes are often disappointing, particularly for serious injuries, and the ongoing loss of function due to poor nerve regeneration is a serious public health problem worldwide. Thus, a successful therapeutic modality to bring functional recovery is urgently needed. With advances in three-dimensional cell culturing, nerve guidance conduits (NGCs) have emerged as a promising strategy for improving functional outcomes. Therefore, they offer a potential therapeutic alternative to nerve autografts. NGCs are tubular biostructures to bridge nerve injury sites via orienting axonal growth in an organized fashion as well as supplying a supportively appropriate microenvironment. Comprehensive NGC creation requires fundamental considerations of various aspects, including structure design, extracellular matrix components and cell composition. With these considerations, the production of an NGC that mimics the endogenous extracellular matrix structure can enhance neuron-NGC interactions and thereby promote regeneration and restoration of function in the target area. The use of electrospun fibrous substrates has a high potential to replicate the native extracellular matrix structure. With recent advances in electrospinning, it is now possible to generate numerous different biomimetic features within the NGCs. This review explores the use of electrospinning for the regeneration of the nervous system and discusses the main requirements, challenges and advances in developing and applying the electrospun NGC in the clinical practice of nerve injuries.

Chlamydia pneumoniae can infect the central nervous system via the olfactory and trigeminal nerves and contributes to Alzheimer's disease risk.

Chlamydia pneumoniae is a respiratory tract pathogen but can also infect the central nervous system (CNS). Recently, the link between C. pneumoniae CNS infection and late-onset dementia has become increasingly evident. In mice, CNS infection has been shown to occur weeks to months after intranasal inoculation. By isolating live C. pneumoniae from tissues and using immunohistochemistry, we show that C. pneumoniae can infect the olfactory and trigeminal nerves, olfactory bulb and brain within 72 h in mice. C. pneumoniae infection also resulted in dysregulation of key pathways involved in Alzheimer's disease pathogenesis at 7 and 28 days after inoculation. Interestingly, amyloid beta accumulations were also detected adjacent to the C. pneumoniae inclusions in the olfactory system. Furthermore, injury to the nasal epithelium resulted in increased peripheral nerve and olfactory bulb infection, but did not alter general CNS infection. In vitro, C. pneumoniae was able to infect peripheral nerve and CNS glia. In summary, the nerves extending between the nasal cavity and the brain constitute invasion paths by which C. pneumoniae can rapidly invade the CNS likely by surviving in glia and leading to Aß deposition.

In vitro modulation of Schwann cell behavior by VEGF and PDGF in an inflammatory environment.

Peripheral glial cell transplantation with Schwann cells (SCs) is a promising approach for treating spinal cord injury (SCI). However, improvements are needed and one avenue to enhance regenerative functional outcomes is to combine growth factors with cell transplantation. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are neuroprotective, and a combination of these factors has improved outcomes in rat SCI models. Thus, transplantation of SCs combined with VEGF and PDGF may further improve regenerative outcomes. First, however, we must understand how the two factors modulate SCs. In this in vitro study, we show that an inflammatory environment decreased the rate of SC-mediated phagocytosis of myelin debris but the addition of VEGF and PDGF (alone and combined) improved phagocytosis. Cytokine expression by SCs in the inflammatory environment revealed that addition of PDGF led to significantly lower level of pro-inflammatory cytokine, TNF-α, but IL-6 and anti-inflammatory cytokines (TGF-ß and IL-10), remained unaltered. Further, PDGF was able to decrease the expression of myelination associated gene Oct6 in the presence of inflammatory environment. Overall, these results suggest that the use of VEGF and/or PDGF combined with SC transplantation may be beneficial in SCI therapy.

Designing accessible educational resources for people living with spinal cord injury.

Context/objective: To identify themes of interest for the production of educational resources for people with spinal cord injury (SCI).Design: A mixed-method study.Setting: Outpatient SCI community in Australia.Participants: Individuals with a SCI, or carers, family & friends of people who live with a SCI (n = 116).Interventions: Not applicable.Outcome measures: Quantify themes of interest perceived within the Australian SCI community as necessary for the development of SCI educational resources.Results: All seven individuals from the focus-group interviews suggested that educational resources on body physiology, secondary complications, injury pathophysiology, and health and wellbeing maintenance would be most pertinent for development. These themes (among others) were further explored and quantitatively evaluated via an online survey which demonstrated that interviewees ranked 'Your injury' as being of highest importance for the production of educational resources. Within each theme, the sub-categories; 'Bowel/bladder' and 'What equipment is covered in the National Disability Insurance Scheme (NDIS)' were ranked as being of highest importance for the production of educational resources.Conclusion: We have identified multiple areas of interest in the design and production of educational resources for individuals with SCI.

Liraglutide modulates olfactory ensheathing cell migration with activation of ERK and alteration of the extracellular matrix.

Transplantation of olfactory ensheathing cells (OECs) is a promising approach for repairing the injured nervous system that has been extensively trialed for nervous system repair. However, the method still needs improvement and optimization. One avenue of improving outcomes is to stimulate OEC migration into the injury site. Liraglutide is a glucagon-like peptide-1 receptor agonist used for management of diabetes and obesity. It has been shown to be neuroprotective and to promote cell migration, but whether it can stimulate glial cells remains unknown. In the current study, we investigated the effects of liraglutide on OEC migration and explored the involved mechanisms. We showed that liraglutide at low concentration (100 nM) overall promoted OEC migration over time. Liraglutide modulated the migratory behavior of OECs by reducing time in arrest, and promoted random rather than straight migration. Liraglutide also induced a morphological change of primary OECs towards a bipolar shape consistent with improved migration. We found that liraglutide activated extracellular signal-regulated kinase (ERK), which has key roles in cell migration; the timing of ERK activation correlated with stimulation of migration. Furthermore, liraglutide also modulated the extracellular matrix by upregulating laminin-1 and down-regulating collagen IV. In summary, we found that liraglutide can stimulate OEC migration and re-model the extracellular matrix to better promote cell migration, and possibly also to become more conducive for axonal regeneration. Thus, liraglutide may improve OEC transplantation outcomes.

Antimicrobial responses of peripheral and central nervous system glia against Staphylococcus aureus.

Staphylococcus aureus infections of the central nervous system are serious and can be fatal. S. aureus is commonly present in the nasal cavity, and after injury to the nasal epithelium it can rapidly invade the brain via the olfactory nerve. The trigeminal nerve constitutes another potential route of brain infection. The glia of these nerves, olfactory ensheathing cells (OECs) and trigeminal nerve Schwann cells (TgSCs), as well as astrocytes populating the glia limitans layer, can phagocytose bacteria. Whilst some glial responses to S. aureus have been studied, the specific responses of different glial types are unknown. Here, we compared how primary mouse OECs, TgSCs, astrocytes and microglia responded to S. aureus. All glial types internalized the bacteria within phagolysosomes, and S. aureus-conjugated BioParticles could be tracked with subtle but significant differences in time-course of phagocytosis between glial types. Live bacteria could be isolated from all glia after 24 h in culture, and microglia, OECs and TgSCs exhibited better protection against intracellular S. aureus survival than astrocytes. All glial types responded to the bacteria by cytokine secretion. Overall, OECs secreted the lowest level of cytokines, suggesting that these cells, despite showing strong capacity for phagocytosis, have immunomodulatory functions that can be relevant for neural repair.

Targeting Matrix Metalloproteinases: A Potential Strategy for Improving Cell Transplantation for Nervous System Repair.

Cell transplantation shows promise for repair of the injured nervous system, including spinal cord injury (SCI) and peripheral nerve injury (PNI). There are, however, still problems hampering these therapies moving from bench to bedside, and the methods need optimization. Three-dimensional (3D) cell culture systems are suggested to improve outcomes, bridging the gap between the in vitro and in vivo environments. In such constructs, cells are allowed to interact with each other and with the extracellular matrix (ECM) in 3D as they do in vivo. Transplanting cells in 3D constructs, rather than in suspension, is thought to promote cell survival and maintain important cellular behaviors. One such critical behavior is cell migration into and within the injury site. Understanding and controlling the migratory capability of 3D-cultured cells is therefore pivotal for developing better transplantation techniques. ECM remodelling can influence numerous cellular functions, including cell migration and matrix metalloproteinases (MMPs) are important enzymes for ECM modulation. Here, we discuss the idea of modulating MMPs to control cell migration in 3D culture systems, which can improve the therapeutic potential of cells transplanted in 3D.

Phagocytosis by Peripheral Glia: Importance for Nervous System Functions and Implications in Injury and Disease.

The central nervous system (CNS) has very limited capacity to regenerate after traumatic injury or disease. In contrast, the peripheral nervous system (PNS) has far greater capacity for regeneration. This difference can be partly attributed to variances in glial-mediated functions, such as axon guidance, structural support, secretion of growth factors and phagocytic activity. Due to their growth-promoting characteristic, transplantation of PNS glia has been trialed for neural repair. After peripheral nerve injuries, Schwann cells (SCs, the main PNS glia) phagocytose myelin debris and attract macrophages to the injury site to aid in debris clearance. One peripheral nerve, the olfactory nerve, is unique in that it continuously regenerates throughout life. The olfactory nerve glia, olfactory ensheathing cells (OECs), are the primary phagocytes within this nerve, continuously clearing axonal debris arising from the normal regeneration of the nerve and after injury. In contrast to SCs, OECs do not appear to attract macrophages. SCs and OECs also respond to and phagocytose bacteria, a function likely critical for tackling microbial invasion of the CNS via peripheral nerves. However, phagocytosis is not always effective; inflammation, aging and/or genetic factors may contribute to compromised phagocytic activity. Here, we highlight the diverse roles of SCs and OECs with the focus on their phagocytic activity under physiological and pathological conditions. We also explore why understanding the contribution of peripheral glia phagocytosis may provide us with translational strategies for achieving axonal regeneration of the injured nervous system and potentially for the treatment of certain neurological diseases.

In vitro cytotoxicity of montelukast in HAPI and SH-SY5Y cells.

Montelukast is a cysteinyl leukotriene (CysLT) receptor antagonist with efficacy against a variety of diseases, including asthma and inflammation-related conditions. However, various neuropsychiatric events (NEs) suspected to be related to montelukast have been reported recently, with limited understanding on their association and underlying mechanisms. This study aimed to investigate whether montelukast can induce neuroinflammation and neurotoxicity in microglial HAPI cells and neural SH-SY5Y cells. The present study also compared the effects of montelukast with a 5-lipoxygenase inhibitor (zileuton) and a cyclooxygenase-2 inhibitor (celecoxib) to better understand modulation of related pathways. HAPI or SH-SY5Y cells were treated with the indicated drugs (3.125 µM-100 µM) for 24 h to investigate drug-induced neuroinflammation and neurotoxicity. Montelukast induced cytotoxicity in HAPI cells (50-100 µM), accompanied with caspase-3/7 activation, prostaglandin E2 (PGE2) release, and reactive oxygen species (ROS) production. Whilst both montelukast and zileuton down-regulated CysLT release in HAPI cells, zileuton did not significantly affect cell viability or inflammatory and oxidative factors. Celecoxib decreased HAPI cell viability (6.25-100 µM), accompanied with increasing caspase-3/7 activation and ROS production, but in contrast to montelukast increased CysLT release and decreased PGE2 production. Similar to observations in HAPI cells, both montelukast and celecoxib (50-100 µM) but not zileuton produced toxicity in SH-SY5Y neuroblastoma cells. Similarly, CM from HAPI cells treated with either montelukast or zileuton produced toxicity in SH-SY5Y cells. The results of the current study show the capability of montelukast to directly induce toxicity and inflammation in HAPI cells, possibly through the involvement of PGE2 and ROS, and toxicity in undifferentiated SH-SY5Y neuroblastoma cells. The current study highlights the importance of consideration between benefit and risk of montelukast usage and provides references for future investigation on decreasing montelukast-related NEs.

Basic insights into Zika virus infection of neuroglial and brain endothelial cells.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain-Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood-brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml-1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.

Transplantation of olfactory ensheathing cells, the glia of the primary olfactory nervous system, has been trialed for spinal cord injury repair with promising but variable outcomes in animals and humans. Olfactory ensheathing cells can be harvested either from the lamina propria beneath the neuroepithelium in the nasal cavity, or from the olfactory bulb in the brain. As these areas contain several other cell types, isolating and purifying olfactory ensheathing cells is a critical part of the process. It is largely unknown how contaminating cells such as fibroblasts, other glial cell types and supporting cells affect olfactory ensheathing cell function post-transplantation; these cells may also cause unwanted side-effects. It is also, however, possible that the presence of some of the contaminant cells can improve outcomes. Here, we reviewed the last decade of olfactory ensheathing cell transplantation studies in rodents, with a focus on olfactory ensheathing cell purity. We analyzed how purification methods and resultant cell purity differed between olfactory mucosa- and olfactory bulb-derived cell preparations. We analyzed how the studies reported on olfactory ensheathing cell purity and which criteria were used to define cells as olfactory ensheathing cells. Finally, we analyzed the correlation between cell purity and transplantation outcomes. We found that olfactory bulb-derived olfactory ensheathing cell preparations are typically purer than mucosa-derived preparations. We concluded that there is an association between high olfactory ensheathing cell purity and favourable outcomes, but the lack of olfactory ensheathing cell-specific markers severely hampers the field.

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, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.