Rapalink-1 Increased Infarct Size in Early Cerebral Ischemia-Reperfusion With Increased Blood-Brain Barrier Disruption

Abstract

It has been reported that the mechanistic target of rapamycin (mTOR) pathway is involved in cerebral ischemia-reperfusion injury. One of the important pathological changes during reperfusion after cerebral ischemia is disruption of blood-brain barrier (BBB). Rapamycin, a first-generation mTOR inhibitor, produces divergent effects on neuronal survival and alteration in BBB disruption. In this study, we investigated how Rapalink-1, a third-generation mTOR inhibitor, would affect neuronal survival and BBB disruption in the very early stage of cerebral ischemia-reperfusion that is within the time window of thrombolysis therapy. The middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Of note, 2 mg/kg of Rapalink-1 or vehicle was administered intraperitoneally 10 min after MCAO. After 1 h of MCAO and 2 h of reperfusion, the transfer coefficient (K_i) of ¹⁴C-α-aminoisobutyric acid (104 Da) and the volume of ³H-dextran (70,000 Da) distribution were determined to assess the degree of BBB disruption. At the same time points, phosphorylated S6 (Ser240/244) and Akt (Ser473) as well as matrix metalloproteinase-2 (MMP2) protein level were determined by Western blot along with the infarct size using tetrazolium stain. Rapalink-1 increased the K_i in the ischemic-reperfused cortex (IR-C, +23%, p < 0.05) without a significant change in the volume of dextran distribution. Rapalink-1 increased the percentage of cortical infarct out of the total cortical area (+41%, p < 0.005). Rapalink-1 significantly decreased phosphorylated S6 and Akt to half the level of the control rats in the IR-C, which suggests that both of the mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2) were inhibited. The MMP2 level was increased suggesting that BBB disruption could be aggravated by Rapalink-1. Taken together, our data suggest that inhibiting both mTORC1 and mTORC2 by Rapalink-1 could worsen the neuronal damage in the early stage of cerebral ischemia-reperfusion and that the aggravation of BBB disruption could be one of the contributing factors.

Keywords: 14C-α-aminoisobutyric acid; Rapalink-1; blood-brain barrer; brain protection; cerebral ischemia-reperfusion; mTOR inhibitor.

Figure 1

Dot plots for the transfer coefficient (K_i) of ¹⁴C-AIB in the examined brain regions of the control group [middle cerebral artery occlusion (MCAO)/reperfusion] and the Rapalink-1 group (MCAO/reperfusion + Rapalink-1) after 1 h of MCAO and 2 h of reperfusion. Each bar represents the mean value of each brain region. A two-way ANOVA followed by the Tukey's test for multiple comparisons was used. The K_i for ¹⁴C-AIB was increased with Rapalink-1 in the IR-C and in the CC. n = 10 in each group. IR-C, ischemic-reperfused cortex; CC, contralateral cortex; IH, ipsilateral hippocampus; CH, contralateral hippocampus; CBLL, cerebellum. *p < 0.05 vs. the control group (MCAO/reperfusion). ^†p < 0.05 vs. pons. ^‡p < 0.05 vs. IR-C.

Figure 2

Dot plots for the volume of dextran distribution in the examined brain regions of the control group (MCAO/reperfusion) and the Rapalink-1 group (MCAO/reperfusion + Rapalink-1) after 1 h of MCAO and 2 h of reperfusion. Each bar represents the mean value of each brain region. A two-way ANOVA followed by the Tukey's test for multiple comparisons was used. There were no significant changes in the volume of dextran distribution with Rapalink-1 in every brain region that was studied. IR-C, ischemic-reperfused cortex; CC, contralateral cortex; IH, ipsilateral hippocampus; CH, contralateral hippocampus; CBLL, cerebellum. n = 10 in each group. ^†p < 0.05 vs. pons.

Figure 3

(A) A diagram with box plots showing the percentage of cortical infarcted area compared with total cortical area in the experimental groups after 1 h of MCAO and 2 h of reperfusion. Each boxplot consists of 25th percentile, median, 75th percentile, and whiskers with the minimum and maximum. Mean is shown with the symbol “×.” Rapalink-1 increased the percentage of the cortical infarct out of the total cortical area. n = 6 in each group. An unpaired Student's t-test was used for the significance. *p < 0.005 vs. the control group (MCAO/reperfusion). (B) Two representative brain sections from the similar brain regions of the control group (MCAO/reperfusion) and the Rapalink-1 group (MCAO/reperfusion + Rapalink-1). The tissue samples were photographed at 2 h after reperfusion without any fixatives. The area of the infarcted cortex appeared pink, not white, which suggests infarction is still going on. The infarcted cortices are marked with arrows. Some of the subcortical areas appeared to be infarcted.

Figure 4

The representative Western blots of pS6, pAkt, and MMP2, and their quantification after 1 h of MCAO and 2 h of reperfusion. ANOVA followed by multiple comparisons with the Bonferroni correction was used for the quantification of protein. The Phosphorylation of Akt at Ser473 and the phosphorylation of S6 at Ser240/244 were increased during ischemia–reperfusion that indicates the increased activity of mTORC2 and mTORC1, respectively. The treatment with Rapalink-1 resulted in a more than 50% decrease in the phosphorylation of both Akt and S6 in the IR-C when compared with the control rats, which suggests that Rapalink-1 inhibited both mTORC1 and mTORC2 in this experiment. The increased protein level of MMP2 with Rapalink-1 treatment in the IR-C suggests that Rapalink-1 could aggravate blood–brain barrier (BBB) disruption in the ischemic-reperfused area. CC, contralateral cortex; IR-C, ischemic-reperfused cortex; Control, MCAO/reperfusion group; Rapalink-1, MCAO/reperfusion + Rapalink-1 group. n = 6–9. *p < 0.05. Values are means ± SD.