Development of a mixed glial in vitro robust platform for therapeutic applications in spinal cord injury

Abstract

Spinal cord injury (SCI) is a catastrophic event, and current strategies for SCI pose several limitations. One of the critical hallmarks of SCI is inflammation, which is due to the activation of microglia and astrocytes. Although there are SCI-related in vitro models relevant to human pathology, most in vitro models focus on studying molecular mechanisms involved in the acute phase. However, there is a lack of assessment of the progression of glial cells-derived inflammation over the acute to the chronic phases. It can be strongly emphasised that understanding this advancement will help establish a framework for evaluating therapeutic strategies in SCI. Therefore, this study aimed for the development and understanding of a model which will be invaluable in facilitating in vitro screening novel therapeutics for the treatment of SCI before proceeding to in vivo evaluation and will provide a platform for high throughput drug screening (HTS).

In phase, 1 of this study, mixed glial cultures (MGCs) consisting of astrocytes, microglia and oligodendrocytes were treated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and interleukin (IL)-6 in combination/s for one day (acute) to 21 days (mimicking acute and chronic inflammation respectively). Lipopolysaccharide (LPS) was used as the positive control. The research unfolds an SCI relevant differential modulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-p65 and mitogen-activated protein kinase (MAPK)-p38 pathways over 21 days, with a unique expression pattern. The expression of these pathways was dependent on the type and combination of cytokines. Among various combinations, a combination of three cytokines was selected for further studies. This treatment resulted in the activation of several pathways involved in secretion of chemokines, pro-inflammatory cytokines, while inhibiting growth factors and anti-inflammatory cytokines mimicking a SCI inflammatory environment in in vivo conditions. Using proteome profiling and ingenuity pathway analysis (IPA) approaches, the causal and comparative network analysis predication highlighted some essential biological functions relevant to SCI immune cell-derived inflammation, a critical factor in this process.

The role of glycans in neuroinflammation and mitochondrial function at chronic stages in the is not yet fully understood. Therefore, phase 2 was focused on studying the response of glycans and mitochondrial function in the inflammatory MGC in vitro model. Lectin microarray and lectin cytochemistry profiling showed glycosylation alterations between different cytokine treatments, LPS and untreated cultures. Lectin cytochemistry demonstrated differential glycosylation in astrocytes and microglia in the mixed population. Lectins co-localised with astrocytes and microglia and indicated higher expression of glycans after cytokine treatment over 21 days. Furthermore, a live-cell metabolic assay platform was used to better understand the bioenergetic phenotype of glia and mitochondrial respiration. Upon cytokine combination treatment, pro-inflammatory glia with increase respiration (also reactive oxygen species), ATP production (by glycolysis), proton leak, and decreased coupling efficiency and membrane potential was observed. This approach has revealed the functionality of mitochondrial activity during inflammation and mimics the pathological condition over seven days.

In the next phase (phase 3), using a cytokine-induced neutrophil chemoattractant (CINC-3) chemokine as a biomarker with an excellent Z’ factor value, a robust HTS platform was established. First, a low volume 384-well plate ELISA method-based detection of CINC-3 was established. Then, using the CINC-3 detection platform, 786 compounds based primary screening was performed and showed the anti-inflammatory effect of selected drugs which further inhibited the production of CINC-3 in secondary screening in dose-dependent manner. Finally, in the fourth phase, to mimic the preclinical or clinical myelin debris (MD)-mediated inflammatory paradigm, a myelin debris-induced mixed glial in vitro model was established. The study showed that the myelin debris induces inflammation in microglia (from MGC), activating the liver X receptor-retinoid X receptor (LXR/RXR) pathway, autophagy, and immune response. Further LXR/RXR agonists could downregulate the heightened immune response and reduce sequestered MD from microglia.

Overall, these results showed that the treatment with cytokine combinations successfully induced acute and chronic-like inflammatory conditions in MGC. An in vitro mixed glial cytokine-induced and myelin debris-induced inflamed model for HTS was established to study the progression of inflammation. These findings have important significance as this approach can be used to investigate novel treatments and conduct drug or molecular efficacy screenings and may replace/enhance preclinical studies for non-prospective drug/compound screening.