Selective Sphingosine 1-Phosphate Receptor 1 Modulation Augments Thrombolysis of Low-Dose Tissue Plasminogen Activator Following Cerebrovascular Thrombosis

Han-Dong Li, Ran Li, Ying Kong, Wenyan Zhang, Caiyun Qi, Dan Wang, Hongying Hao, Qiang Liu, Han-Dong Li, Ran Li, Ying Kong, Wenyan Zhang, Caiyun Qi, Dan Wang, Hongying Hao, Qiang Liu

Abstract

Background: Results from our recent study demonstrate that sphingosine-1-phosphate receptor 1 (S1PR1) modulation improves microvascular hemodynamics after cerebrovascular thrombosis. This study was to determine the microvascular hemodynamic effects of a sub-thrombolytic dose of tPA combined with a selective S1PR1 modulator ozanimod in a mouse model of cerebrovascular thrombosis.

Methods: Microvascular circulation in mice was monitored in vivo by two-photon microscopy. Thrombosis was induced in cortical arterioles by laser irradiation. Arteriolar flow velocity was measured by line-scanning following plasma-labeling with fluorescein-dextran.

Results: Laser-induced thrombosis led to a persistent reduction of flow velocity in cortical arterioles. Sub-thrombolytic dose of tPA along with vehicle control did not improve the flow velocity in cortical arterioles following thrombosis. In contrast, a sub-thrombolytic dose of tPA along with ozanimod dramatically restored flow velocity in cortical arterioles following thrombosis. Ozanimod did not affect coagulation and bleeding time. Notably, ozanimod reduced thrombus volume without altering microvascular lumen diameter. In addition, ozanimod reduced leukocyte components within the thrombus.

Conclusions: These findings demonstrate that the thrombolytic effect of a sub-thrombolytic dose of tPA is markedly enhanced by S1PR1 modulation, implying that S1PR1 modulation may improve the therapeutic benefit of low-dose tPA in patients with acute ischemic stroke.

Trial registration: ClinicalTrials.gov NCT02002390.

Keywords: ischemic stroke; microcirculation; tPA; thrombolysis; two-photon microscopy.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Li, Li, Kong, Zhang, Qi, Wang, Hao and Liu.

Figures

Figure 1
Figure 1
Effect of thrombosis on flow velocity in cortical arterioles measured by 2‐photon laser scanning microscopy. (A) Images and diagrams show the induction of thrombosis in cortical arterioles using 2‐photon laser scanning microscopy. (B) Z-stack Images show cortical arterioles at baseline, 60 min after thrombosis and up to 90 min after vehicle treatment. 3D images show horizontal and vertical views of thrombus. The yellow lines indicate the horizontal and vertical cross sections of thrombus. White arrows indicate the direction of blood flow. White dots and numbers indicate individual location to measure flow velocity. Scale bar: 50 μm in thrombus images, 30 μm in cross sections. Line scans were performed to measure flow velocity along the longitudinal axis of arterioles of interest (flow velocity is expressed as mm per second). The slopes (Δx/Δt) of the measurement-angles are proportional to flow velocity. Scale bar: 40 μm. Time bar: 0.1 sec. (C) Line graph shows thrombus volume at 60 min after thrombosis, 30 min and 90 min after vehicle treatment. (D) Flow velocity at indicated time points. n = 10 per group. Mean ± SD.
Figure 2
Figure 2
Effect of low-dose tPA on flow velocity in cortical arterioles following thrombosis. (A) Z-stack Images show cortical arterioles and flow velocity at baseline, 60 min after thrombosis and up to 90 min after vehicle plus low-dose tPA (5 mg/kg, i.v.) treatment. 3D images show horizontal and vertical views of thrombus. The yellow lines indicate the horizontal and vertical cross sections of thrombus. White arrows indicate the direction of blood flow. White dots and numbers indicate individual location to measure flow velocity. Scale bar: 50 μm in thrombus images, 30 μm in cross sections. Line scans were performed to measure flow velocity along the longitudinal axis of arterioles of interest (flow velocity is expressed as mm per second). The slopes (Δx/Δt) of the measurement-angles are proportional to flow velocity. Scale bar: 40 μm. Time bar: 0.1 sec. (B) Line graph shows thrombus volume at 60 min after thrombosis, 30 min and 90 min after tPA + vehicle treatment. (C) Flow velocity at indicated time points. n = 10 per group. Mean ± SD.
Figure 3
Figure 3
Effect of ozanimod and low-dose tPA treatment on flow velocity in cortical arterioles following thrombosis. (A) Z-stack Images show cortical arterioles and flow velocity at baseline, 60 min after thrombosis and up to 90 min after ozanimod (0.6 mg/kg, i.v.) plus low-dose tPA (5 mg/kg, i.v.) treatment. 3D images show horizontal and vertical views of thrombus. The yellow lines indicate the horizontal and vertical cross sections of thrombus. White arrows indicate the direction of blood flow. White dots and numbers indicate individual location to measure flow velocity. Scale bar: 50 μm in thrombus images, 30 μm in cross sections. Line scans were performed to measure flow velocity along the longitudinal axis of arterioles of interest (flow velocity is expressed as mm per second). The slopes (Δx/Δt) of the measurement-angles are proportional to flow velocity. Scale bar: 40 μm. Time bar: 0.1 sec. (B) Line graph shows thrombus volume at 60 min after thrombosis, 30 min and 90 min after tPA + Ozanimod treatment. **p<0.01 versus 60 min after thrombosis. (C) Flow velocity at indicated time points. n = 10 per group. Mean ± SD. **p<0.01 versus vehicle + tPA group.
Figure 4
Figure 4
Effect of ozanimod on coagulation and bleeding time. (A) Bar graph shows coagulation time in mice receiving vehicle, ozanimod (0.6 mg/kg, i.v.), 5 mg/kg tPA or ozanimod (0.6 mg/kg) + 5 mg/kg tPA. (B) Bar graph shows bleeding time in mice receiving vehicle, ozanimod (0.6 mg/kg, i.v.), 5 mg/kg tPA or ozanimod (0.6 mg/kg) + 5 mg/kg tPA. n = 6 per group. Mean ± SD.
Figure 5
Figure 5
Effect of ozanimod on thrombus volume and lumen diameter in cortical arterioles following thrombosis. (A, B). Z-stack Images show thrombus at 60 min after thrombosis and 90 min after ozanimod (0.6 mg/kg, i.v.) or vehicle treatment. 3D images show horizontal and vertical views of thrombus. The yellow lines indicate the horizontal and vertical cross sections of thrombus. Scale bar: 50 μm in thrombus images, 30 μm in cross sections. (C). Bar graph shows the changes of thrombus volume at 90 min after ozanimod or vehicle treatment. Volume change = thrombus volume at 90 min after treatment - baseline volume at 30 min after thrombosis. n = 8 per group. (D). Bar graph shows the changes of lumen diameter at 90 min after ozanimod or vehicle treatment. Lumen diameter was calculated at 60 min after thrombosis and 90 min after ozanimod or vehicle treatment. n = 8 per group. Mean ± SD. *p < 0.05.
Figure 6
Figure 6
Effect of ozanimod on the composition of thrombus in cortical arterioles. Brain sections were prepared from brain tissues harvested at 90 min after ozanimod (0.6 mg/kg, i.v.) or vehicle treatment. (A) Z-stack immunostaining images show CD3+ T cells (green) within Dextran Texas-Red labeled thrombus in cortical arterioles. (B) Bar graph shows the counts of CD3+ T cells within thrombus. (C) Z-stack immunostaining images show CD45+ cells (green) within Dextran Texas-Red labeled thrombus. (D) Bar graph shows the counts of CD45+ cells (green) within thrombus. Scale bar: 40 μm. n = 6 per group. Mean ± SD. **p < 0.01.

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