Background

Astrocytes and microglial cells play a crucial role during the development, as well as in the normal physiology and pathology of the nervous system. These include structural, vascular, metabolic and neuro-regulatory functions like modulation of transmission by forming ‘quad-partite synapses’, release of gliotransmitters and trophic factors, and integration of neuronal-glial networks (Parpura et al., 1994; Araque et al., 1999; Schafer et al., 2013; Michell-Robinson et al., 2015; Rossi, 2015). Microglia, in addition to the canonical immune functions, also plays an important role in adult neurogenesis, differentiation, maturation and integration within the neuronal circuitry (Rezaie and Male, 2002; Polazzi and Monti, 2010; Michell-Robinson et al., 2015; Ransohoff and El Khoury, 2015). In response to a pathological insult, the glial cells react by undergoing morphological and physiological changes and rescue the vulnerable neurons from the insult (Ferraiuolo et al., 2011; Pekny et al., 2014; Mishra et al., 2016; Mishra et al., 2017). At the tissue level, astrocytes undergo morphological transformation from protoplasmic to fibrillary phenotype and move to the site of injury to create a physical barrier between damaged and healthy cells, or by forming glial scars in a process called reactive gliosis. In a similar manner, microglia undergo morphological transformation from the resting/ramified phenotype to the activated/amoeboid phenotype, via intermediate activation stages, (Rezaie and Male, 2002; Michell-Robinson et al., 2015; Ransohoff and El Khoury, 2015). Both the glial cell types thus promote neuronal repair through acute inflammation, which modulates the microenvironment by regulating production and release of trophic factors, gliotransmitters, cytokines and chemokines (Raivich et al., 1999; Parpura et al., 2012; Verkhratsky and Butt, 2013; Pekny et al., 2014). Reactive gliosis is reversible and beneficial during the acute insult. However, in response to prolonged insult or genetic dysregulation, the balance is disrupted and may lead to irreversible and erroneous activation resulting in chronic inflammation and neurodegeneration (Lobsiger and Cleveland, 2007). Therefore, delineating the precise glial response as well as their role in modulating neuronal physiology becomes relevant. However, an in vivo approach may present with a disadvantage owing to the complex neuron-glial and glial-glial interactions, which dynamically modulate these changes at the tissue level. Primary glial cultures help us to overcome this issue, by enabling observations in an isolated cell-type model system. In vitro experimentation also widens the scope for further comparing the effect of neuron-glial/glial-glial interactions through a co-culture model system.

Various approaches have been adopted to culture astrocytes and microglia from animal brains and spinal cords. These methods vary considerably not only in terms of purity and yield, but also with respect to the cell population targeted for isolation (Giulian and Baker, 1986; Saura et al., 2003; Floden and Combs, 2007; Scorisa et al., 2010; Kerstetter and Miller, 2012). For instance, the conventional repeated shaking methods for culturing microglia exploit the microglial layer growing atop astrocytes in the mixed glial cultures (Giulian and Baker, 1986; Scorisa et al., 2010), while the mild trypsinization method targets the ones growing beneath (Saura et al., 2003). We compared all the methods and standardized a procedure to establish exceedingly enriched astroglial and microglial cultures, while maximizing the yield. The current protocol generates enough microglial and astroglial cells per spinal cord to execute parallel experiments in both the cell types, thus minimizing the inter-population variability while comparing cell-specific responses.