Remote postconditioning ameliorates stroke damage by preventing let-7a and miR-143 up-regulation

Antonio Vinciguerra, Pasquale Cepparulo, Serenella Anzilotti, Ornella Cuomo, Valeria Valsecchi, Salvatore Amoroso, Lucio Annunziato, Giuseppe Pignataro, Antonio Vinciguerra, Pasquale Cepparulo, Serenella Anzilotti, Ornella Cuomo, Valeria Valsecchi, Salvatore Amoroso, Lucio Annunziato, Giuseppe Pignataro

Abstract

Remote limb ischemic postconditioning (RLIP) is a well-established neuroprotective strategy able to protect the brain from a previous harmful ischemic insult through a sub-lethal occlusion of the femoral artery. Neural and humoral mechanisms have been proposed as mediators required to transmit the peripheral signal from limb to brain. Moreover, different studies suggest that protection observed at brain level is associated to a general genetic reprogramming involving also microRNAs (miRNAs) intervention. Methods: Brain ischemia was induced in male rats by transient occlusion of the middle cerebral artery (tMCAO), whereas RLIP was achieved by one cycle of temporary occlusion of the ipsilateral femoral artery after tMCAO. The expression profile of 810 miRNAs was evaluated in ischemic brain samples from rats subjected either to tMCAO or to RLIP. Among all analyzed miRNAs, there were four whose expression were upregulated after stroke and returned to basal level after RLIP, thus suggesting a possible involvement in RLIP-induced neuroprotection. These selected miRNAs were intracerebroventricularly infused in rats subjected to remote ischemic postconditioning, and their effect was evaluated in terms of brain damage, neurological deficit scores and expression of putative targets. Results: Twenty-one miRNAs, whose expression was significantly affected by tMCAO and by tMCAO plus RLIP, were selected based on microarray microfluidic profiling. Our data showed that: (1) stroke induced an up-regulation of let-7a and miR-143 (2) these two miRNAs were involved in the protective effects induced by RLIP and (3) HIF1-α contributes to their protective effect. Indeed, their expression was reduced after RLIP and the exogenous intracerebroventricularly infusion of let-7a and miR-143 mimics prevented neuroprotection and HIF1-α overexpression induced by RLIP. Conclusions: Prevention of cerebral let-7a and miR-143 overexpression induced by brain ischemia emerges as new potential strategy in stroke intervention.

Keywords: let-7a; miR-143; microRNA; remote limb postconditioning; stroke.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

© The author(s).

Figures

Figure 1
Figure 1
Heat map of microRNA expression profiles in the whole ischemic area from sham-operated, tMCAO and tMCAO + RLIP animals. (A) The average of signal intensity values for each significantly expressed miRNA is reported (p < 0.05). Red and green indicate up- and down-regulation respectively, according to the colorimetric scale in the right side of the panel. a, b and c indicate the three experimental group: (a) Sham, (b) tMCAO and (c) tMCAO + RLIP animals. Each vertical line refers to three samples for each experimental group. (B) Volcano plot analysis in the lower side of panel shows the comparison of miRNA levels after tMCAO and RLIP induction. The y-axis corresponds to the mean expression value of log10 (p-value), and the x-axis displays the log2 fold change value. The blue dots represent the miRNA levels expressed in rat brain 24 h after stroke induction; the orange dots represent the miRNA levels expressed in rat brain 24 h after tMCAO + RLIP.
Figure 2
Figure 2
Validation of microarray results by real-time PCR. MicroRNA levels analysed by Real-Time PCR in ischemic brain regions from rats subjected to tMCAO and tMCAO + RLIP are expressed as fold change over the respective sham-operated controls. Each column represents the mean ± S.E.M. Results of microRNAs expression were normalized with respect to 4.5S RNA as internal control. n = 3 or 4 per group. S is for Sham-operated group.
Figure 3
Figure 3
In vivo effect of miRNAs administrations on ischemic volume and neurological scores of rats subjected to Remote Limb Ischemic Postconditioning or to tMCAO. (A) Ischemic damage was assessed on rats subjected to continuous icv infusion of miRNA mimic (0,6 mg/kg) or Anti-miR (9 µg/kg) from 24 h before RLIP induction up to 24 h after reperfusion. On the top, representative pictures of brain sections comprising cortex and striatum are included. Brain damage is in white. Each column represents the mean ± S.E.M. n = 6 rats per tMCAO + RLIP + negative CTL group; n = 5 rats per tMCAO + RLIP + let-7a group; n = 6 rats per tMCAO + RLIP + Anti-let-7a group; n = 5 rats per tMCAO + RLIP + miR-143 group; n = 6 rats per tMCAO + RLIP + Anti-miR-143 group; n = 4 rats per tMCAO + RLIP + miR-485 group; n = 5 rats per tMCAO + RLIP + miR-451 group. *: p < 0,05 Vs tMCAO + RLIP + Negative CTL mimic. Dashed line indicates infarct volume referred to tMCAO animals. (B-C) Measurement of general and focal scores of neurological deficits of rats subjected to continuous icv infusion of miRNA mimic (0,6 mg/kg) or Anti-miR (9 µg/kg) from 24 h before tMCAO and RLIP induction up to 24 h after reperfusion. Each column represents the mean ± S.E.M. n = 6 rats per tMCAO + RLIP + negative CTL group; n = 5 rats per tMCAO + RLIP + let-7a group; n = 6 rats per tMCAO + RLIP + Anti-let-7a group; n = 5 rats per tMCAO + RLIP + miR-143 group; n = 6 rats per tMCAO + RLIP + Anti-miR-143 group; n = 4 rats per tMCAO + RLIP + miR-485 group; n = 5 rats per tMCAO + RLIP + miR-451 group. *: p < 0,05 Vs tMCAO + RLIP + Negative CTL mimic. (D) Ischemic damage was assessed on rats subjected to continuous icv infusion of miRNA mimic (0,6 mg/kg) or Anti-miR (9 µg/kg) from 24 h before tMCAO induction up to 24 h after reperfusion (Figure 4D). On the top, representative pictures of brain sections comprising cortex and striatum are included. Brain damage is in white. Each column represents the mean ± S.E.M. n = 5 rats per tMCAO + negative CTL group; n = 4 rats per tMCAO + let-7a group; n = 6 rats per tMCAO + Anti-let-7a group; n = 5 rats per tMCAO + miR-143 group; n = 6 rats per tMCAO + Anti-miR-143 group; *: p < 0,05 Vs tMCAO + negative CTL mimic. (E-F) Measurement of general and focal scores of neurological deficits of rats subjected to continuous icv infusion of miRNA mimic (0,6 mg/kg) or Anti-miR (9 µg/kg) from 24 h before tMCAO induction up to 24h h after reperfusion. Each column represents the mean ± S.E.M. n = 5 rats per tMCAO + negative CTL group; n = 4 rats per tMCAO + let-7a group; n = 6 rats per tMCAO + Anti-let-7a group; n = 5 rats per tMCAO + miR-143 group; n = 6 rats per tMCAO + Anti-miR-143 group; *: p < 0,05 Vs tMCAO + negative CTL mimic.
Figure 4
Figure 4
Effect of Anti-let-7a administration on ischemic volume and neurological scores of rats subjected to tMCAO after 7 days of reperfusion. (A) Ischemic damage was assessed on rats subjected to continuous icv infusion of Anti-miR (9 µg/kg) from 24 h before tMCAO induction up to 24 h after reperfusion and sacrificed 7 days after ischemia induction. On the top, representative pictures of brain sections comprising cortex and striatum are included. Brain damage is in white. Each column represents the mean ± S.E.M. n = 5 rats per tMCAO + negative CTL group; n = 5 rats per tMCAO + Anti-let-7a group; n = 5 rats per tMCAO + RLIP + negative CTL. (B-C) Measurement of general and focal scores of neurological deficits of rats subjected to continuous icv infusion of Anti-miR (9 µg/kg) from 24 h before tMCAO and tMCAO induction up to 24 h after reperfusion. Each column represents the mean ± S.E.M. n = 5 rats per tMCAO + negative CTL group; n = 5 rats per tMCAO + Anti-let-7a group; n = 5 rats per tMCAO + RLIP + negative CTL.
Figure 5
Figure 5
Effect of 100 min of transient brain ischemia (tMCAO) and RLIP on miR-143 expression in cortical ischemic region. (A-I-M-U) Confocal microscopic images displaying NeuN and GFAP in red, (B-J-N-V) Mir143-3p in green, (C-K-O-W) Hoechst in blue and (D-L-P-X) Merge in yellow in the brain ischemic regions of rats subjected to Sham-operation, tMCAO and tMCAO + RLIP. A representative brain slice cartoon indicating the area of interest is on the top of the Figure. Scale bars in A-L 75 μm in M-X 50 μm. High magnification zoom 2x in pictures H-T.
Figure 6
Figure 6
Effect of 100 min of transient brain ischemia (tMCAO) and RLIP on let-7a expression in cortical ischemic region. (A-I, M-U) Confocal microscopic images displaying NeuN and GFAP in red, (B-J-N-V) let-7a in green, (C-K-O-W) Hoechst in blue and (D-L-P-X) Merge in yellow in the brain ischemic regions of rats subjected to Sham-operation, tMCAO and tMCAO + RLIP. A representative brain slice cartoon indicating the area of interest is on the top of the Figure. Scale bars in A-L 75 μm in M-X 50 μm. High magnification zoom 2x in pictures H-T.
Figure 7
Figure 7
Evaluation of HIF1α protein expression in tMCAO + RLIP rats upon mimic-let-7a-5p administration. Evaluation of HIF-1α protein expression in ischemic rats subjected to remote limb postconditioning, intracerebroventricularly infused with mimic-let-7a-5p and sacrificed at 24 h from reperfusion. Protein levels are expressed as percentage versus the sham-operated controls. Each column represents the mean ± S.E.M. Results of protein expression were normalized with respect to α-tubulin. On the top of each graph, representative blots of HIF1α and α-tubulin signals are shown (A) HIF1α protein levels in cortex (n = 4 samples per sham group; n = 5 samples per tMCAO and RLIP groups; n = 4 samples per mimic-let-7a group). *: p < 0.05 vs. sham-operated controls.

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