Impact of 12/15-lipoxygenase on brain injury after subarachnoid hemorrhage

Abstract

Background and Purpose

Subarachnoid hemorrhage (SAH) is a devastating form of stroke. Oxidative stress contributes to brain injury, but the mechanisms have been poorly studied. Here, we evaluated the role of 12/15-lipoxygenase (12/15-LOX), an enzyme known to cause cell death in ischemic stroke, on brain injury in a mouse model of SAH.

Methods

C57Bl6 wild-type mice and ALOX15 knockout mice were subjected to SAH using a direct blood injection technique. In SAH wild-type mice, half received the 12/15-LOX inhibitor ML351, and half received vehicle. Immunohistochemistry, brain edema, blood-brain barrier (BBB) leakage and functional outcomes were assessed 1 and 3 days after SAH induction.

Results

SAH led to increased 12/15-LOX in macrophages of the brain parenchyma, adjacent to the subarachnoid blood. Neuronal cell death after SAH was reduced by ML351 and in ALOX15 knockout mice. Similarly, SAH induced brain edema which was 12/15-LOX dependent. Finally, ALOX15 gene knockout, and inhibitor treatment in WT mice with SAH led to an improved behavioral outcome.

Conclusions

12/15-LOX is overexpressed in macrophages after SAH in mice, and inhibition of the 12/15-LOX pathway decreases brain injury and improves neurological outcome. This study suggests 12/15-LOX as a novel therapeutic target to limit brain injury after SAH.

Introduction

Aneurysmal subarachnoid hemorrhage (SAH) is a severe form of stroke, which often leads to death and disability.¹ Two phenomena lead to brain injury: early brain injury (EBI) and delayed cerebral ischemia (DCI), frequently associated with cerebral vasospasm.² EBI refers to the effect of transient global ischemia and toxicity of subarachnoid blood, causing apoptotic neuronal cell death through several mechanisms.³ Mechanisms involved in EBI persist for several days, and neuroprotection against EBI has to be developed.⁴ 12/15-Lipoxygenase (12/15-LOX), an enzyme involved in the oxidative pathway, has been identified as a key target to prevent secondary brain injury after ischemic stroke.^5,6,7 Here, we evaluated the role of 12/15-LOX in EBI after SAH, and the impact of a highly specific 12/15-LOX inhibitor, ML351.⁸

Materials and Methods

Data supporting the findings of this study are available from the corresponding authors upon reasonable request. Detailed Materials and Methods are deposited in an online supplementary file (please see http://stroke.ahajournals.org). Animal experiments were performed following protocols approved by the MGH Institutional Animal Care and Use Committee in accordance with NIH Guidelines. Briefly, SAH was induced in 11 ALOX15 knockout mice and 80 genetically matched wild-type mice using an established direct blood injection technique. I.p. injection of the 12/15-LOX inhibitor ML351 (50 mg/kg)⁸ or vehicle occurred five minutes after induction of SAH. Immunohistochemistry was assessed 1 and 3 days later; and brain edema, BBB leakage and functional outcomes were assessed 3 days later. The flow-chart of the study is described in supplementary figure I.

Results

12–15-LOX is overexpressed in macrophages after SAH

Blood was directly injected into the chiasmatic cistern to establish SAH (Fig 1A). Following sacrifice one day later, immunohistochemistry revealed that 12/15-LOX is overexpressed in SAH mice compared to sham-operated mice (Fig 1B). This overexpression was restricted to the brain parenchyma adjacent to the SAH. Cells expressing 12/15-LOX are CD68+, suggesting a central role for activated macrophages in 12/15-LOX overexpression (Fig 1C, Sup Fig II). Neither neurons nor astrocytes expressed 12/15-LOX 24h after SAH (Sup Fig III-IV).

Fig. 1: 12/15-LOX is overexpressed after subarachnoid hemorrhage.

(A) Schematic view representing the needle trajectory for SAH induction, and the field of view used for the histologic experiments. CA: carotid artery; ON: Optic nerve. (B) Representative immunohistochemistry images obtained 24 hours after SAH induction, and corresponding quantification (n= 4 per groups). SAH induces a major increase of 12/15-LOX compared to sham mice.Scale bar, 50 µm; *p<0,05 versus Sham (C) Representative immunohistochemistry images obtained 24 hours after SAH induction, showing that the cells expressing 12/15-LOX are CD68+, so are activated macrophages. Scale bar, 50 µm. (D) Representative immunohistochemistry images obtained 72 hours after SAH induction, and corresponding quantification (n= 4 per groups). SAH still induce an increase of 12/15-LOX compared to sham mice. This phenomenon is partly reversed by the 12/15-LOX inhibitor ML351, and absent in 12/15-LOX ko mice. Scale bar, 50 µm; *p<0,05 versus Sham; **p<0,05 versus SAH+vehicle.

12/15-LOX overexpression increases brain injury after SAH

To investigate the impact of 12/15-LOX on brain injury, we induced SAH in ALOX15 knockout mice, or injected the 12/15-LOX inhibitor ML351 to wild-type mice with SAH. 12/15-LOX expression remained slightly elevated in wild-type mice 3 days after SAH, which was reduced by ML351 treatment, whereas ALOX15 knockout mice exhibited only background staining for 12/15-LOX (Fig 1D). Next we detected widespread neuronal cell death surrounding the SAH area, as assessed by Fluorojade B staining in SAH mice, compared to ALOX15 knockout mice and mice receiving ML351 (Fig 2A-B). Moreover, SAH mice developed cerebral edema, which was reduced in ALOX15 knockout mice, but not by ML351 (Fig 2C). We did not detect BBB leakage in this model (Fig 2D), suggesting that edema in this model is mainly cytotoxic, not vasogenic.

Fig. 2: 12/15-LOX plays a key role in neuronal cell death and brain edema after SAH.

(A) Representative pictures of FJB staining performed 72 hours after SAH induction. SAH induces widespread FJB staining indicative of neuronal damage, a phenomenon limited in 12/15-LOX ko mice or in mice receiving the 12/15-LOX inhibitor ML351. ON: optic nerve; CA: carotid artery. Scale bar, 200 µm. (B) Corresponding quantification (n=4 per group). *p<0,05 versus Sham; **p<0,05 versus SAH+vehicle. (C) Brain edema measured using the dry/wet weight method in both hemispheres three days after SAH induction. SAH induces brain edema, which is reduced in 12/15-LOX ko mice (n=10 in WT mice groups, and 7 in the 12/15-LOX ko mice group). *p<0,05 versus Sham; **p<0,05 versus SAH+vehicle; NS: non-significant versus SAH+vehicle. (D) No opening of the BBB is detected in the 2 brain hemispheres with Evans Blue (EB) (n=7 in Sham and SAH+vehicle group, n=9 in SAH+ML351 group). NS: non-significant versus Sham.

12/15-LOX inhibition improves the short term functional outcome after SAH

This model of SAH induces short-term neurological impairments. SAH-associated weight loss was reduced in ALOX15 knockout mice (Fig 3A). Using a 4-point neuroscore, we found that the neurological deficit caused by SAH is decreased in mice receiving ML351 (Fig 3B). Little injury was detected using a modified Garcia scale (Fig 3C). Finally, spontaneous motor activity was decreased in SAH mice, which was reversed by ML351 and in ALOX15 knockout mice (Fig 3D).

Fig. 3: Inhibition of 12/15-LOX improves functional outcomes 3 days after SAH.

(A) SAH leads to significant weight loss. This phenomenon is reversed in 12/15-LOX ko mice, and decreased in animals treated with ML351 (n=20 in Sham and SAH+ML351 mice, n=18 in SAH+Vehicle mice, n=11 in 12/15-LOX ko mice). *p<0,05 versus Sham; **p<0,05 versus SAH+vehicle; NS : non-significant versus SAH+vehicle. (B) The 12/15-LOX inhibitor ML351 improves neurological deficit after SAH, evaluated by a 4-point neuroscore scale (n=20 in the WT mice groups, n=11 in the 12/15-LOX ko mice group). *p<0,05 versus Sham; **p<0,05 versus SAH+vehicle; NS: non-significant versus SAH+vehicle. (C) No significant deficit in a modified Garcia scale (n=20 in the WT mice groups, n=11 in the 12/15-LOX ko mice group). NS: non-significant versus Sham. (D) Spontaneous activity is better preserved in mice treated with ML351 compared to vehicle following SAH, and in 12/15-LOX ko mice (n=20 in the WT mice groups, n=11 in the 12/15-LOX ko mice group). *p<0,05 versus Sham; **p<0,05 versus SAH+vehicle.

Discussion

In our study, 12/15-LOX was overexpressed in activated macrophages after SAH, and blocking 12/15-LOX activity decreased the level of brain injury and subsequently improved the short-term neurological outcome in mice.

This result is in line with previous studies evaluating the role of 12/15-LOX in ischemic stroke: 12/15-LOX is upregulated in the peri-infarct area in mice and humans, contributing to brain damage by causing both neuronal cell death, and neurovascular injury.^5,6 Surprisingly, 12/15-LOX is here increased predominantly in macrophages at 24h, rather than in neurons and endothelial cells; the injury mechanism may thus differ. Fewer 12/15-LOX positive cells are present at 72h. Considering the number of CD68+ cells after SAH is stable within the first 7 days after SAH,⁹ this suggests its expression is transient. The enzyme inhibitor ML351 further reduced the amount of 12/15-LOX, possibly due to the vicious cycle of amplification of oxidative stress caused by 12/15-LOX.⁸

The role of oxidative stress in SAH has long been known.¹⁰ Interestingly, baicalein administration in rats with SAH decreased EBI, notably because baicalein is a 12/15-LOX inhibitor.¹¹ Baicalein as a strong antioxidant is not very specific however, and data focusing strictly on 12/15-LOX were needed, particularly since the 12/15-LOX product 12-HETE is elevated in the CSF of SAH patients.¹²

Our study has several limitations. First, our study only looked at short term outcomes relevant to EBI and found fewer neurological deficits than previously; a possible impact of 12/15-LOX on cerebral vasospasm and DCI remains to be explored. Additionally, we have used male mice, and effects of 12/15-LOX in females remain to be determined. Moreover, future studies are needed to examine the mechanisms of 12/15-LOX injury in more detail, focusing on its activity in macrophages.