In the era of three-dimensional (3D) imaging, standard immunohistochemistry has been recognized as highly limited in studies of pathology and complex human anatomy. By analyzing only narrow slices, larger structures and essential aspects of tissue architecture may lose critical contextual details, leading to incomplete or misleading interpretations. While two-dimensional (2D) sections have supported a basic understanding of histoarchitecture, they are insufficient for capturing the true three-dimensional reality of biological tissues. Increasing numbers of studies across multiple anatomical regions now adopt techniques that isolate intact anatomical units - flat mount, whole-mount, or en face preparations - combined with high-resolution fluorescent microscopy. These approaches offer a more comprehensive visualization and interpretation of structural organization. This is particularly significant in the olfactory nervous system, where defining complex cellular mechanisms underlying its remarkable regenerative ability requires a full-thickness context. Thin sections risk missing key pathophysiological features that remain poorly defined. Given these limitations and the knowledge gap surrounding the cellular interactions driving olfactory regeneration, developing a refined whole-mount technique capable of preserving full-thickness tissue complexity was essential. Entire olfactory mucosae were isolated as full-thickness samples, accompanied by an optimized antibody-penetration protocol. High-resolution imaging (EVIDENT SpinSR) and 3D reconstruction enabled accurate visualization of cellular interactions in vivo.

Spinal cord injury (SCI) is a debilitating condition resulting in a range of neurological impairments up to complete loss of function below the level of injury. With current clinical management limited to decompression and stabilisation of the injury, there is urgent need to develop effective restorative treatments. In animal models, cell transplantation therapies are being tested that utilise different cell types including olfactory ensheathing cells (OECs), a type of glial cell, to support and promote regeneration. While OECs have a unique combination of properties highly suitable for SCI repair, their efficacy and consistency need to be improved. Evidence suggests a combinational approach using growth factors or compounds alongside OECs may stimulate their innate properties and alter the internal milieu of an injury site in favour of neural repair. Naturally, there is intricate interplay between various growth factors and OECs during development of the olfactory system, and in injury and repair events, which regulate their migration, phagocytosis, and proliferation. Therefore, exploiting different growth factors to selectively enhance OECs’ therapeutic potential could lead to restorative treatment of SCI. While some studies have already explored using growth factors to treat SCI in animal models, an optimal ‘cocktail’ has yet to be identified. In seeking to identify such a cocktail, this review presents the current understanding of SCI and the therapeutic potential of OECs and explores combined use of growth factors and OECs to improve treatment outcomes.

Background & AimSpinal cord injury is a debilitating condition attributed to the damage of neuronal connections in the nervous system. There is currently no cure but research has presented cell therapies as a promising strategy. Olfactory Mucosal Olfactory Ensheathing Cells (OM-OECs), a glial cell population that maintains neural connectivity in the olfactory nervous system is a feasble candidate due to its safety, accessibility and reparative properties. OECs derived from the OM contain other cell types and since patient outcomes are varied, standardisation of a transplantation cell therapy will need a well-defined population of cells.Here we present a comparison of three different techniques to enrich OECs from the olfactory mucosa of a genetically modified S100β-DsRed mice.Methods, Results & Conclusion: Method 1Enrichment of OECs using differential detachment propertiesOM derived cells were cultured using an enzymatic dissociation method. These cells were then subject to two rounds of trypsinisations at 3 minute intervals to allow for the detachment of cells.Results 1An enriched population that contained 56.27+/- 0.87% DsRed+ OECs after one round of dissociation, and 61% +/- 5.21 DsRed+ OECs after two rounds were obtained.Method 2Enrichment of OECs using compliment lysis (CL) of CD90 positive fibroblastsHere we attempted to enrich OECs using compliment mediated lysis of fibroblasts. The cell specific compliment activating CD90 antibody was used to achieve depletion of cellsResult 2There was 50-70% reduction in CD90 positive fibroblasts however, other cells in culture compensated for this loss, producing no significant enrichment of the OECs.Method 3Enrichment of OECs using selective adhesion to microcarriersMicrocarriers have been used for expansion of OM derived cells. Here we show that OECs and associated cells have varied adhesion for different microcarriers.Of serveral microcarriers tested OECs show favoured attachment to Hillex II microcarriers allowing for its enrichment.Result 3Enrichment of DsRed+ OECs upto 3 fold was obtained with variying results across replicates.ConclusionOur results show that the differential detachment method is the most suitable method for the purification of OECs for producing sufficient quantities of cells enriched with OECs. The CL method depletes CD90+ cells but other compensating cells prevent OEC enrichment. Lastly, Hillex II beads could be used for the expansion of an OEC enriched population of OM cells with further modifications.

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 β1-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.

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-β 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.

>One of the key challenges in neuroscience is that the central nervous system(CNS; the brain and spinal cord), is largely unable to regene rate after injury. One factor contributing to this lack of repair is the accumulation of cellular and myelin debris at the site of injury. The debris is not efficiently phagocytosed and can persist for years after the initial injury, resulting in an inflammatory environment which inhibits axonal regrowth(Lutz and Barres, 2014).

Neurotrauma injuries are notoriously difficult to deal with both clinically as well as experimentally, as the cellular and molecular events ensuing after injury complicate the neuroinflammatory processes. Spinal cord injuries are further complicated by the formation of scars at the injury sites, which can provide a physical barrier to repair. The lack of effective clinical therapy for spinal cord injury underscores the need for experimental approaches to generate effective therapies. To repair the injury, cell transplantation offers the potential to replace lost cells and create a permissive bridge to promote neural regeneration across the injury site. Olfactory ensheathing cells (OECs), which are the glia of the olfactory nerve, stand apart from other candidate cell types due to their innate natural abilities to manage nerve injury and promote repair and regeneration. This is evidenced by their physiological role in the daily repair and maintenance of the olfactory nerve. Here, we explain their properties in relation to their physiological role and their most relevant cellular attributes, including cellular interactions, phagocytosis, migration, axonal guidance and support, and modulation of neuroinflammation. We highlight some critical drawbacks in the current approaches and identify some ways to address them.

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.

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.

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.

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.

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.

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 .

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.

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 Aβ deposition.

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.

James St John grew up in Melbourne, Australia and started his scientific career in agricultural science [...]

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.

Welcome to Neuroglia, a new Open Access MDPI journal which will report original research articles and reviews on studies of neuroglia [...]