The ensuing imbalance between excitatory and inhibitory transmission is the primary pathogenic mechanism for cell death in stroke (Costa C, et al. 2004), and inhibiting the excitotoxicity protects against the brain damage. Numerous inhibitors of glutamatergic transmission have been tested as neuroprotective agents following experimental stroke, but none has been shown to be of clinical value (Green AR, et al. 2000). GABA is the primary inhibitory neurotransmitter in the mammalian brain and has been demonstrated to counteract excessive glutamatergic excitation, thereby protecting neurons from excitotoxicity during stroke (Galeffi F, et al. 2000). However, many compounds shown to increase GABA function do not possess robust neuroprotective efficacy in focal stroke models (Green AR, et al. 2000).
Slit-Robo signaling regulates the development of interneuron populations in cerebral cortex (Andrews W, et al. 2008; Marín O and Rubenstein JL. 2003), we demonstrate that Slit 2 overexpression can increase peri-infarct GABAergic activity, which was protective in focal microstroke. It was reported that curcumin upregulate Slit 2 expression (Sirohi VK, et al 2017), which protect against stroke-mediated brain damage (Bhat A, et al. 2019). Curcumin is widely consumed as turmeric (Bavarsad K, et al. 2019), which may be a potential treatment to upregulate GABAergic activity around microinfarcts, a possibility that we will study in further study.
In addition, Slit 2 was reported to reduce inflammatory responses following ischemic insults (Altay T, et al. 2007). Consistent with these findings, overexpression of Slit 2 inhibited local microglial and astrocytic activation and protected against peri-infarct neuronal loss. Slit 2 is expressed in multiple brain cell types, including neurons, astrocytes, endothelial cells (Wu JY, et al. 2001), and pericytes (Guijarro-Muñoz I, et al. 2012), and different ischemic models induce distinct cell type-specific changes in Slit 2 expression. For instance, Park et al. (Park JH, et al. 2016) reported that Slit 2 was constitutively expressed in neurons of control rats and that transient forebrain ischemia upregulated Slit 2 ligand in reactive astrocytes but not neurons or activated microglia of the hippocampus. Fang et al. (Fang M, et al. 2010) reported that Slit 2 was expressed mainly by neurons of the temporal lobe during the acute and latent phases of temporal lobe epilepsy (TLE) in rats, but mainly in astrocytes during the chronic phase. In our study, Slit 2 was expressed in both cortical neurons and astrocytes but not in microglia of control mice, while MI increased astrocytic expression and reduced neuronal expression. Slit-Rob signaling is high in interneurons, so reduced in Slit 2 expression likely reflected loss of peri-infarct interneurons and ensuing local excitotoxicity. Indeed, Slit 2 overexpression protected against interneuronal loss and reduced excitotoxic sequela.
Slit 2 was reported to be overexpressed in these Tg mice throughout life (Han HX, et al. 2011, Li JC, et al. 2018; Li G, et al. 2018), which promoted paravascular clearance (Li G, et al. 2018). Consistent with these findings, we demonstrated that microstroke impaired AQP 4 function, paravascular clearance (Gaberel T, et al. 2014), and BBB permeability (Chen X, et al. 2018), all of which were protected by Slit 2 overexpression. Finally, we demonstrated that Slit 2 overexpression promoted neuronal plasticity after cortical microinfarct induction. Slit 2 has been reported to promote both axonal elongation and branching (Ma L and Tessier-Lavigne M, 2007) or dendritic growth and branching in developing cortical cells (Whitford KL, et al. 2002), as well as GABAergic function in mature brain. Collectively, these processes contribute to circuit recovery post-ischemia. Indeed, GABA-mediated inhibition is a critical modulator of cortical remapping, which is required for functional recovery after stroke (Hiu T, et al. 2016). It is worthy of note that parietal cortex contributes to route learning using proximal salient cues in the water maze task (Solari N, et al. 2018). Thigmotaxis (swimming alone the tub edge) is indicative of spatial learning failure (Vorhees CV, et al. 2006) and we observed thigmotaxis in WT MIs mice, but not Slit 2-Tg MI mice, suggesting that Slit 2 overexpression protected against the spatial learning impairment induced by parietal microinfarcts.
In summary, we demonstrate that Slit 2 overexpression can preserve the excitatory–inhibitory balance in peri-infarct regions of parietal cortex by promoting GABAergic transmission, thereby protecting against excitotoxicity and local neuroinflammation. Furthermore, Slit 2 overexpression protected against glymphatic system and BBB dysfunction, attenuated local neuronal loss, and ultimately prevented cognitive decline induced by parietal microinfarcts.