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[Preprint]. 2024 Jun 12:rs.3.rs-4468292.
doi: 10.21203/rs.3.rs-4468292/v1.

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi after Subarachnoid Hemorrhage in Mice

Ari Dienel  1 , Sung Ha Hong  1 , Hussein A Zeineddine  1 , Sithara Thomas  1 , C M Shafeeque  1 , Dania A Jose  1 , Kiara Torres  1 , Jose Guzman  1 , Andrew Dunn  2 , T P Kumar  1 , Gadiparthi N Rao  3 , Spiros L Blackburn  1 , Devin W McBride  1
Affiliations

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi after Subarachnoid Hemorrhage in Mice

Ari Dienel et al. Res Sq. .

Update in

Abstract

Background and purpose: Impaired cerebral circulation, induced by blood vessel constrictions and microthrombi, leads to delayed cerebral ischemia after subarachnoid hemorrhage (SAH). 12/15-Lipooxygenase (12/15-LOX) overexpression has been implicated in worsening early brain injury outcomes following SAH. However, it is unknown if 12/15-LOX is important in delayed pathophysiological events after SAH. Since 12/15-LOX produces metabolites that induce inflammation and vasoconstriction, we hypothesized that 12/15-LOX leads to microvessel constriction and microthrombi formation after SAH, and thus 12/15-LOX is an important target to prevent delayed cerebral ischemia.

Methods: SAH was induced in C57BL/6 and 12/15-LOX-/- mice of both sexes by endovascular perforation. Expression of 12/15-LOX was assessed in brain tissue slices and in vitro. C57BL/6 mice were administered either ML351 (12/15-LOX inhibitor) or vehicle. Mice were evaluated for daily neuroscore and euthanized on day five to assess cerebral 12/15-LOX expression, vessel constrictions, platelet activation, microthrombi, neurodegeneration, infarction, cortical perfusion, and for development of delayed deficits. Finally, the effect of 12/15-LOX inhibition on platelet activation was assessed in SAH patient samples using a platelet spreading assay.

Results: In SAH mice, 12/15-LOX was upregulated in brain vascular cells and there was an increase in 12-S-HETE. Inhibition of 12/15-LOX improved brain perfusion on days 4-5 and attenuated delayed pathophysiological events, including microvessel constrictions, microthrombi, neuronal degeneration, and infarction. Additionally, 12/15-LOX inhibition reduced platelet activation in human and mouse blood samples.

Conclusions: Cerebrovascular 12/15-LOX overexpression plays a major role in brain dysfunction after SAH by triggering microvessel constrictions and microthrombi formation, which reduces brain perfusion. Inhibiting 12/15-LOX may be a therapeutic target to improve outcomes after SAH.

Keywords: 12/15-Lipooxygenase; arterioles; delayed neurological deficit; microthrombi; microvessel constrictions; platelets; subarachnoid hemorrhage.

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Conflict of interest statement

Potential conflict of interests The authors declare no competing interests.

Figures

Figure 1:
Figure 1:
Expression of 12/15-LOX. (A-C) Immunostaining for 12- and 15-LOX in brains from male mice 5 days after SAH. 12/15-LOX is green, laminin (vasculature) is red. Scale Bar = 50 μm. (D) Quantification of 12-LOX protein. n=6/group/sex. One-way ANOVA with Tukey. *p<0.05 vs. Sham, #p<0.05 vs. SAH D1. (E) Protein expression (Western blot) of 12- and 15-LOX and β-Actin in human brain pericytes, human brain microvascular endothelial cells, and mouse brain microvascular endothelial cells.
Figure 2
Figure 2. Plasma 12-S-HETE levels after SAH.
n=5/group/sex. Kruskal-Wallis with Dunn’s test. *p<0.05 vs Sham, #p<0.05 vs SAH D1, §p<0.05 vs SAH D5.
Figure 3:
Figure 3:. 12/15-LOX inhibition reduces plasma 12-S-HETE on post-SAH Day 5.
(A) Male mice. (B) Female mice. n=5/group/strain/sex, except for female Sham + Veh which n=4. One-way ANOVA test with Tukey (male) and Dunn’s multiple comparison test (female), unpaired t-test for 12/15-LOX−/− mice. *p<0.05 vs Sham + Veh or Sham, #p<0.05 vs SAH + Veh. One data point for the Sham + Veh group was excluded since it was more than 7800% from the mean.
Figure 4:
Figure 4:. 12/15-LOX inhibition reduces microvessel constrictions on post-SAH Day 5.
(A) Representative images of microvessel constrictions in the brain. (B, C) Quantification of constrictions in arterioles. n=6–8/group/sex. One-way ANOVA with Tukey post-hoc. *p<0.05 vs Sham + Veh, #p<0.05 vs SAH + Veh. Unpaired t-test for 12/15-LOX−/− mice: §p<0.05 vs 12/15-LOX−/−-Sham.
Figure 5:
Figure 5:. 12/15-LOX inhibition reduces platelet activation after SAH in mice and humans.
(A) Representative images of mouse platelet morphology. Scale bar = 5μm. (B-C) Quantification of platelet activation for mice. One-way ANOVA with Tukey post-hoc. n=6/group. *p<0.05 vs Sham + Veh and #p<0.05 vs SAH + Veh. (D) Quantification of platelet activation for SAH patients. Lines show the mean values of platelet activation from control patient’s blood treated with vehicle (black dashed) or ML351 (red dotted). Unpaired t-test. n=5–9/group/time-point. #p<0.05.
Figure 6:
Figure 6:. 12/15-LOX inhibition reduces microthrombi on Day 5 after SAH.
(A) Representative image of microthrombi in the brain of a SAH mouse. Scale bar: 50μm. (B, C) Quantification of microthrombi in mice. n=6/group/sex/strain. One-way ANOVA with Tukey post-hoc. *p<0.05 vs Sham + Veh. Unpaired t-Test for 12/15-LOX−/− mice.
Figure 7:
Figure 7:. 12/15-LOX inhibition with ML351 reduces neurodegeneration on Day 5 after SAH.
(A) Representative image of neurodegeneration. (B-E) Quantification of neuronal degeneration in the brain cortex (B, C) and striatum (D, E). n=6/group/sex/strain. One-way ANOVA with Tukey post-hoc. *p<0.05 vs Sham + Veh, #p<0.05 vs SAH + Veh. Unpaired t-test for 12/15-LOX−/− mice: §p<0.05 vs 12/15-LOX−/−-Sham.
Figure 8:
Figure 8:. Inhibition of 12/15-LOX improves brain perfusion in the watershed area.
n=6/group/sex/strain. Two-way ANOVA with Sidak’s multiple comparison test. * p<0.05 vs SAH + Veh.
Figure 9:
Figure 9:. 12/15-LOX inhibition ameliorates neurological deficits in SAH mice.
n=25–41 male/group/time-point and n=25–30 female/group/time-point. Friedman ANOVA followed by Wilcoxon signed-rank posthoc then Bonferroni correction. *p<0.05 vs Sham + Veh vs SAH + Veh, #p<0.05 vs Sham + Veh vs SAH + ML351, †p<0.05 vs SAH + Veh vs SAH + ML351.
Figure 10
Figure 10. DND incidence.
Data from all mice of the same sex were combined for DND analysis. SAH and SAH + Vehicle male mice were combined to increase power since there was no difference in DND incidence between these two injury control groups (Supplemental Fig 6). Log-rank (Mantel-Cox) Test.
Figure 11:
Figure 11:. Day 5 infarct area after SAH.
n=6–8/group/sex/strain. One-way ANOVA with Tukey post-hoc. *p<0.05 vs Sham + Veh, #p<0.05 vs SAH + Veh. Unpaired t-test for 12/15-LOX−/− mice: §p<0.05 vs 12/15-LOX−/−-Sham.
Figure 12
Figure 12
Schematic of the role of 12/15-Lipooxygenase in inducing microthrombi and microvessel constrictions after SAH.
See this image and copyright information in PMC

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