Increased Expression of Tight Junction Proteins and Blood-Brain Barrier Integrity in MCAO Rats Following Injection of miR-149-5p

Abstract

Cerebral ischemia is a common neurodegenerative disease in which damage to the blood-brain barrier (BBB) is the main consequence. In cerebral ischemia, the level of miR-149-5p and tight junction proteins are decreased, while the level of Calpine is increased, finally leading to increased BBB permeability. This study investigated the effect of miR-149-5p mimic on the expression of Calpain, Occludin, and ZO-1 and the consequences of cerebral ischemia. Cerebral ischemia model was performed via middle cerebral artery occlusion (MCAO) method on female Wistar rats. Four groups of Wistar rats were studied: Sham, cerebral ischemia without treatment, Scramble miR, and miR-149-5p mimic treatment. Then, neurological defects and BBB permeability (via Evans blue staining), cerebral edema (cerebrospinal fluid percentage), and ZO-1, Occludin, and Calapin expression (by quantitative real time- PCR) were investigated. qRT-PCR results showed miR-149-5p expression decreases after cerebral ischemia induction. In addition, Occludin and ZO-1 expression significantly increased in miR-149-5p group. In contrast, Calapin expression, BBB permeability, brain water content and neurological defects were significantly decreased. It seems that the increased level of miR-149-5p exerts its protective effect on cerebral ischemia due to increasing of tight junction proteins.

Keywords: Cerebral ischemia; miR-149-5p; middle cerebral artery occlusion; tight junction.

Fig.1

Study timeline

Fig.2

Changes of miR-149-5p following cerebral ischemia. Induction of cerebral ischemia led to a decrease in the level of miR-149-5p in the damaged hemisphere. The level of this miR in the model group showed a significant difference compared to the sham group (P<0.001 The injection of miR mimic led to a considerable increase in the level of miR-149-5p compared to the model group (P<0.001). S-miR injection did not affect increasing miR levels. The data are expressed as the mean ±SD. ***P<0.001 vs. Sham group, ^###P<0.001 vs. MCAO Model and NC Groups. (n=8).

Fig.3

Comparison of neurological defects in the studied groups. Cerebral ischemia led to movement disorders in rats, and miR-149-5p mimic group significantly reduced neurological defects in miR mimic group rats compared to the model group (P<0.001). However, S-miR injection did not significantly change the score of neurological defects. The data are expressed as the mean ±SD. **P<0.01 vs. Sham group, ^##P<0.01 vs. MCAO Model and NC Groups. (n=8)

Fig.4

Brain samples containing EB in the affected hemisphere. (A) Comparison chart of EB concentration in different groups (B). and Comparing the absorption of the samples obtained from the brain hemispheres of rats with the standard graph showed that following the induction of cerebral ischemia, the concentration of EB in the damaged hemisphere increased, and there was a significant difference in the concentration of EB in the healthy and damaged hemispheres (P=0.000). The injection of miR mimic caused a considerable decrease in EB concentration in the damaged hemisphere compared to the damaged hemisphere of the model group (P<0.001). In contrast, the injection of S-miR did not have a significant effect. There was no significant difference in the concentration of EB in the healthy hemispheres of different groups (P=0.000). The data are expressed as the mean ±SD. ***P<0.001 vs. Sham group, ^##P<0.01 vs. NC Group, ^&&P<0.01 vs. MCAO Model, ^$$$P<0.001 vs. Opposite hemisphere in the same group. (n=8).

Fig.5

Comparison of brain water content in the studied groups. An increase in cerebrospinal fluid content occurred after cerebral ischemia. The injection of miR-149-5p mimic showed a significant decrease in the percentage of cerebrospinal fluid (P<0.001). There was no significant difference in brain water content between the right hemispheres of the studied groups. The data are expressed as the mean ±SD. ***P<0.001 vs. Sham group, ###P<0.001 vs. MCAO Model and NC Groups, $$$P<0.001 vs. Opposite hemisphere in the same group. (n=8).

Fig.6

Comparison of ZO-1 and Occludin levels in the studied groups. Following the induction of ischemia, ZO-1 and Occludin showed a significant decrease in the damaged hemisphere of rats, and a significant difference between the levels of these two genes was revealed in the model and sham groups (P=0.003). The injection of miR mimic caused a considerable increase in the level of ZO-1 and Occludin compared to the model group (P=0.004). In contrast, the injection of S-miR did not affect the level of these two genes compared to the model group. The data are expressed as the mean ±SD. **P<0.01 vs. Sham group, ###P<0.001 vs. MCAO Model and NC Groups. (n=8)

Fig.7.

Calpain level changes in the studied groups. Cerebral ischemia led to a significant increase in the Calpain level in the model group compared to the sham group (P=0.000), and the treatment of rats with miR-149-5p mimic led to a rise in its level in the miR mimic group compared to the model group (P<0.001). Treatment with S-miR did not affect Calpain downregulation. The data are expressed as the mean ±SD. ***P<0.001 vs. Sham group, ###P<0.001 vs. MCAO Model and NC Groups. (n=8).