Ischemic Preconditioning Modulates the Peripheral Innate Immune System to Promote Anti-Inflammatory and Protective Responses in Mice Subjected to Focal Cerebral Ischemia

The development of tolerance triggered by a sublethal ischemic episode (preconditioning, PC) involves a complex crosstalk between neurons, astrocytes and microglia, although the role of the peripheral immune system in this context is largely unexplored. Here, we report that severe cerebral ischemia caused by transient middle cerebral artery occlusion (MCAo) in adult male mice elevates blood counts of inflammatory neutrophils and monocytes, and plasma levels of miRNA-329-5p. These inflammatory responses are prevented by ischemic PC induced by 15 min MCAo, 72h before the severe insult (1h MCAo). As compared with sham-operated animals, mice subjected to either ischemic PC, MCAo or a combination of both (PC+MCAo) display spleen contraction. However, protein levels of Ym1 (a marker of polarization of myeloid cells towards M2/N2 protective phenotypes) are elevated only in spleen from the experimental groups PC and PC+MCAo, but not MCAo. Conversely, Ym1 protein levels only increase in circulating leukocytes from mice subjected to 1h MCAo, but not in preconditioned animals, which is coincident with a dramatic elevation of Ym1 expression in the ipsilateral cortex. By immunofluorescence analysis, we observe that expression of Ym1 occurs in amoeboid-shaped myeloid cells, mainly representing inflammatory monocytes/macrophages and neutrophils. As a result of its immune-regulatory functions, ischemic PC prevents elevation of mRNA levels of the pro-inflammatory cytokine interleukin (IL)-1β in the ipsilateral cortex, while not affecting IL-10 mRNA increase induced by MCAo. Overall, the elevated anti-inflammatory/pro-inflammatory ratio observed in the brain of mice pre-exposed to PC is associated with reduced brain infarct volume and ischemic edema, and with amelioration of functional outcome. These findings reaffirm the crucial and dualistic role of the innate immune system in ischemic stroke pathobiology, extending these concepts to the context of ischemic tolerance and underscoring their relevance for the identification of novel therapeutic targets for effective stroke treatment

Modulation of Cerebral Store-operated Calcium Entry-regulatory Factor (SARAF) and Peripheral Orai1 Following Focal Cerebral Ischemia and Preconditioning in Mice

Store-operated Ca2+ entry (SOCE) contributes to Ca2+ refilling of endoplasmic reticulum (ER), but also provides Ca2+ influx involved in physiological and pathological signalling functions. Upon depletion of Ca2+ store, the sensor protein stromal interaction molecule (STIM) activates Orai1, forming an ion-conducting pore highly selective for Ca2+. SOCE-associated regulatory factor (SARAF) associates with STIM1 to facilitate a slow form of Ca2+-dependent inactivation of SOCE or interacts with Orai1 to stimulate SOCE in STIM1-independent manner. We have investigated whether cerebral ischemic damage and neuroprotection conferred by ischemic preconditioning (PC) in mouse are associated with changes in the expression of the molecular components of SOCE. Ischemic PC induced by 15-min occlusion of the middle cerebral artery (MCAo) resulted in significant amelioration of histological and functional outcomes produced, 72 h later, by a more severe ischemia (1 h MCAo). Neither ischemia, nor PC affected the expression of Orai1 in the frontoparietal cortex. However, the number of Orai1-immunopositive cells, mostly corresponding to Ly-6G+ neutrophils, was significantly elevated in the blood after the ischemic insult, regardless of previous PC. The expression of Stim1 and SARAF, mainly localised in NeuN-immunopositive neurons, was reduced in the ischemic cortex. Interestingly, neuroprotection by ischemic PC prevented the reduction of SARAF expression in the lesioned cortex and this could be interpreted as a compensatory mechanism to restore ER Ca2+ refilling in neurons in the absence of STIM1. Thus, preventing SARAF downregulation may represent a pivotal mechanism implicated in neuroprotection provided by ischemic PC and should be exploited as an original target for novel stroke therapies