Brain Ischemic Tolerance Triggered by Preconditioning Involves Modulation of Tumor Necrosis Factor-α-Stimulated Gene 6 (TSG-6) in Mice Subjected to Transient Middle Cerebral Artery Occlusion

Abstract

Ischemic preconditioning (PC) induced by a sub-lethal cerebral insult triggers brain tolerance against a subsequent severe injury through diverse mechanisms, including the modulation of the immune system. Tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, has recently been involved in the regulation of the neuroimmune response following ischemic stroke. Thus, we aimed at assessing whether the neuroprotective effects of ischemic PC involve the modulation of TSG-6 in a murine model of transient middle cerebral artery occlusion (MCAo). The expression of TSG-6 was significantly elevated in the ischemic cortex of mice subjected to 1 h MCAo followed by 24 h reperfusion, while this effect was further potentiated (p < 0.05 vs. MCAo) by pre-exposure to ischemic PC (i.e., 15 min MCAo) 72 h before. By immunofluorescence analysis, we detected TSG-6 expression mainly in astrocytes and myeloid cells populating the lesioned cerebral cortex, with a more intense signal in tissue from mice pre-exposed to ischemic PC. By contrast, levels of TSG-6 were reduced after 24 h of reperfusion in plasma (p < 0.05 vs. SHAM), but were dramatically elevated when severe ischemia (1 h MCAo) was preceded by ischemic PC (p < 0.001 vs. MCAo) that also resulted in significant neuroprotection. In conclusion, our data demonstrate that neuroprotection exerted by ischemic PC is associated with the elevation of TSG-6 protein levels both in the brain and in plasma, further underscoring the beneficial effects of this endogenous modulator of the immune system.

Keywords: TSG-6; cerebral ischemia; neuroprotection; preconditioning; stroke.

Figure 1

Transient MCAo and ischemic PC modulate plasma levels of TSG-6 protein and of miR-23a, miR-23b, and miR-744. (A) TSG-6 protein, (B) miR-23a, (C) miR-23b, or (D) miR-744 relative expression levels in plasma of mice who have undergone SHAM surgery (SHAM), PC (15 min MCAo), transient (1 h) MCAo, or PC + MCAo. * p < 0.05 vs. SHAM, ** p < 0.01 vs. PC and p < 0.001 vs. MCAo, # p < 0.001 vs. SHAM (one-way ANOVA followed by Tukey’s post-test, n = 6–8 mice per experimental group).

Figure 2

Ischemic PC potentiates elevation of TSG-6 protein expression in the brain of mice subjected to transient MCAo. (A) TSG-6 protein expression levels detected by Western blotting in the ipsilateral (ischemic, I) and contralateral (C) cortex of mice subjected to SHAM surgery (SHAM), PC (15 min MCAo), transient (1 h) MCAo, or PC + MCAo (** p < 0.01 vs. SHAM ipsilateral, vs. PC ipsilateral and vs. corresponding contralateral, * p < 0.05 vs. PC + MCAo ipsilateral (two-way ANOVA followed by Bonferroni post-test, n = 6–8 animals per experimental group). (B) Representative immunofluorescence images of the ipsilateral frontal cortex showing colocalization (yellow overlapping in merge panels) of TSG6 (green fluorescence) with GFAP-immunopositive neurons (red fluorescence). (C) Representative immunofluorescence images of the ipsilateral parietal cortex showing colocalization (yellow overlapping in merge panels) of TSG-6 (green fluorescence) with Ly6B.2-immunopositive myeloid cells (i.e., granulocytes and monocytes/macrophages, red fluorescence). Nuclei were counterstained with DAPI (blue signal). Scale bars = 150 μm.

Figure 3

Ischemic PC reduces cerebral infarct volume caused by transient MCAo in mice. (A) Representative cresyl violet-stained coronal brain slices and (B) infarct damage expressed as percentage of ipsilateral hemisphere of mice subjected to 1 h MCAo followed by 24 h reperfusion, preceded (PC + MCAo) or not (MCAo) by 15 min MCAo, 72 h before. *** p < 0.0001 vs. MCAo (Student’s t test, n = 8 animals per experimental group).