Background

Microglia are resident immune cells of the central nervous system (CNS), which continuously scan their environment for pathologic alterations (Nimmerjahn et al., 2005). Upon detection of such conditions, microglia transform into an activated state and migrate towards the source where they perform different tasks (Kreutzberg, 1996; Davalos et al., 2005). Pathologic alterations, such as neurodegenerative diseases or acute injuries, are associated with neuronal loss and phagocytosis of these dying cells by activated microglia. Therefore, the overall assumption is that microglia are involved in this process (Thanos, 1991; Davalos et al., 2005; Hanisch and Kettenmann, 2007). Besides a possible effect of microglia on dying neurons, microglia are also part of the glial scar, which is formed at the lesion site and impairs axon regeneration in the CNS (Silver, 2004; Kitayama et al., 2011). However, as a clear discrimination of infiltrated blood born macrophages and microglia in the injured nerve was not feasible, the specific role of microglia in glial scar formation and axonal regeneration remained elusive (Silver, 2004; Hanisch and Kettenmann, 2007; Prinz and Priller, 2014; Hilla et al., 2017).

A suitable model to address the role of microglia in CNS degeneration and axonal regeneration is the visual pathway with its retinal ganglion cells (RGCs), whose axons project through the optic nerve (Leibinger et al., 2009; Fischer and Leibinger, 2012). Upon axonal damage, axons normally fail to regenerate (Thanos, 1991; Fischer et al., 2000). The degenerative and regenerative processes, following acute injury, can be easily visualized in retinal wholemounts and optic nerves, respectively as presented in the current protocol. Furthermore, the visual system and particularly RGCs are a suitable model to study neurodegenerative processes in chronic diseases such as multiple sclerosis or glaucoma (Kerrison et al., 1994; Howell et al., 2007; Diekmann and Fischer, 2013; Dietrich et al., 2018). In addition, the optic nerve is widely used as a classical model to study general regenerative failure in the CNS and strategies to facilitate axon growth (Fischer and Leibinger, 2012).

Several studies have been published, suggesting detrimental effects of microglia on RGC survival and axon regeneration whereas other studies indicate a beneficial, neuroprotective role (Thanos et al., 1993; Levkovitch-Verbin et al., 2006; Bosco et al., 2008; Chen et al., 2012; Rice et al., 2015). However, due to a paucity of depletion methods, many studies initially analyzed the contribution of microglia by altering their activation state with pharmacologic approaches (Thanos et al., 1993; Levkovitch-Verbin et al., 2006; Bosco et al., 2008; Jiao et al., 2014). In recent years, genetic methods were developed to achieve microglia depletion (Heppner et al., 2005; Bruttger et al., 2015; Waisman et al., 2015). However, apart from the requirement of transgenic animals, these methods have potential drawbacks such as the development of astrogliosis, secretion of pro-inflammatory cytokines and blood-brain-barrier damage, which complicate data interpretation. Only recently, pharmacologic colony stimulating factor 1 receptor (CSF1R) inhibitors PLX3397 and PLX5622 have been developed, which allow near complete depletion of microglia in the CNS independent of the animals genetic background (Elmore et al., 2014; Spangenberg et al., 2016; Hilla et al., 2017). In fact, we have recently demonstrated that an oral PLX5622 treatment for 21 days leads to a complete depletion of microglia in the retina and almost a total elimination in the optic nerve (Hilla et al., 2017). In accordance with previous studies, this approach selectively induces apoptosis of microglia by CSF1R inhibition, while other phagocytic cells such as peripheral macrophages are not affected (Elmore et al., 2014; Hilla et al., 2017). Although some studies suggested that this treatment causes minor astrocyte activation in the brain, we did not find any in the optic nerve or retina (Elmore et al., 2014; Hilla et al., 2017). Furthermore, as the inhibitor is orally bioavailable and capable of passing the blood brain barrier, PLX5622 can be formulated in standard rodent chow preventing the necessity of regular injections. Thus, this approach allows scientists to unequivocally address the role of microglia for different pathologic CNS paradigms, such as injury or chronic diseases, not only in the visual system, but also in other prominent CNS tissues such as brain and spinal cord. Using this approach in the visual pathway, we found that microglia neither affect the degeneration process of RGCs nor their capability to regenerate injured axons into the optic nerve upon an acute injury (Hilla et al., 2017). The current protocol describes the method for depleting microglia in the visual system and how effects on acute degeneration of RGCs and axon regeneration in the optic nerve can be quantified to identify the contribution of microglia in this context. This protocol can be easily expanded to investigate the involvement of microglia in other paradigms of acute and chronic injury or diseases in the visual system.