The inhibitor of 20-HETE synthesis, TS-011, improves cerebral microcirculatory autoregulation impaired by middle cerebral artery occlusion in mice

Abstract

Background and purpose: 20-Hydroxyeicosatetraenoic acid is a potent vasoconstrictor that contributes to cerebral ischaemia. An inhibitor of 20-Hydroxyeicosatetraenoic acid synthesis, TS-011, reduces infarct volume and improves neurological deficits in animal stroke models. However, little is known about how TS-011 affects the microvessels in ischaemic brain. Here, we investigated the effect of TS-011 on microvessels after cerebral ischaemia.

Experimental approach: TS-011 (0.3 mg·kg(-1) ) or a vehicle was infused intravenously for 1 h every 6 h in a mouse model of stroke, induced by transient occlusion of the middle cerebral artery occlusion following photothrombosis. The cerebral blood flow velocity and the vascular perfusion area of the peri-infarct microvessels were measured using in vivo two-photon imaging.

Key results: The cerebral blood flow velocities in the peri-infarct microvessels decreased at 1 and 7 h after reperfusion, followed by an increase at 24 h after reperfusion in the vehicle-treated mice. We found that TS-011 significantly inhibited both the decrease and the increase in the blood flow velocities in the peri-infarct microvessels seen in the vehicle-treated mice after reperfusion. In addition, TS-011 significantly inhibited the reduction in the microvascular perfusion area after reperfusion, compared with the vehicle-treated group. Moreover, TS-011 significantly reduced the infarct volume by 40% at 72 h after middle cerebral artery occlusion.

Conclusions and implications: These findings demonstrated that infusion of TS-011 improved defects in the autoregulation of peri-infarct microcirculation and reduced the infarct volume. Our results could be relevant to the treatment of cerebral ischaemia.

Figure 1

A) Representative coronal section of mouse brain stained with 2,3,5-triphenyltetrazolium chloride. The imaged area we focused by two-photon laser-scanning microscopy is indicated by the yellow bar. The adjacent, less stained cortex was the area infarcted at 72 h after reperfusion. B) Representative two-photon laser-scanning microscopy image of cerebral microvessels at a depth of 75 µm, which have been stained with SR101. The regions of interest for the measurement of the blood flow velocities in the microvessels are circled and numbered. C) SR101 intensity curves for the regions of interest of each microvessel shown in B. The AUC was calculated within the range of the decay curve indicated in this graph. The SR101 intensity was expressed as an arbitrary unit (A.U.).

Figure 2

A) Representative two-photon laser-scanning microscopy images of cerebral microvessels at a depth of 75 µm, which have been stained with SR101. The regions of interest for the measurements by the method using the SR101 decay curve and the line-scan method are indicated by the circle (AUC, left panel) and the line (Line-scan, right panel) respectively. B) (i) Representative SR101 intensity curve within the regions of interest. (ii) Representative line-scan image showing the linear shadows produced by negatively stained red blood cells. The blood flow velocity was calculated as the slope (Δx/Δt). C) Relation between the middle cerebral artery occlusion-induced reductions in blood flow velocities as measured by the method using the SR101 decay curve (AUC) and the line-scan method. A statistically significant correlation between the two methods was observed using Pearson product–moment correlations.

Figure 3

Effect of TS-011 on blood flow velocities in a mouse transient middle cerebral artery occlusion (MCAO) model. TS-011 (0.3 mg·kg⁻¹) or the vehicle was intravenously administered as a 1 h infusion at 0 (immediately after reperfusion), 6, 12 and 18 h after reperfusion. The values for the blood flow velocities during MCAO, 1, 2, 4, 7 and 24 h after reperfusion are expressed as a percentage of the corresponding control value obtained before MCAO (100%). The times of the TS-011 infusions are shown by the black bars. The numbers in parentheses indicate the number of vessels studied per group. ***P < 0.001, significantly different from the control value (Student's t-test with Bonferroni correction for multiple comparisons); ###P < 0.001, ##P < 0.01, significantly different from the vehicle-treated group (Student's t-test).

Figure 4

Effect of TS-011 on the microvascular perfusion area after reperfusion in a mouse transient middle cerebral artery occlusion model. Representative binarized z-stacked images before (corresponding to 1 h after reperfusion) and 120 min after treatment with the vehicle or TS-011 are shown in the upper panels. TS-011 (0.3 mg·kg⁻¹) or the vehicle was administered as a 1 h infusion after the first scanning. The values for the microvascular perfusion area at various time points are expressed as a percentage of the corresponding control value obtained at first scanning (100%). The differences between the two time-courses was analysed with a two-way repeated measures anova; *P < 0.05, significantly different from the vehicle-treated group. The numbers in parentheses indicate the number of z-stacked images analysed per group.

Figure 5

Effect of TS-011 on infarct volume in a mouse transient middle cerebral artery occlusion model. Representative images of the 2,3,5-triphenyltetrazolium chloride-stained sections are shown in the upper panels. The infarct volumes were determined using 2,3,5-triphenyltetrazolium chloride-staining at 72 h after reperfusion. TS-011 (0.3 mg·kg⁻¹) or the vehicle was administered as a 1 h infusion every 6 h until 72 h after reperfusion. *P < 0.05 significantly different from the vehicle-treated group (Student's t-test). The numbers in parentheses indicate the number of animals studied per group.