Ultra-early cerebral thrombosis formation after experimental subarachnoid hemorrhage detected on T2* MRI

Abstract

Background and Purpose:

The mechanisms of brain damage during ultra-early subarachnoid hemorrhage (SAH) have not well studied. The current study examined the SAH-induced hyperacute brain damage at 4 hours using magnetic resonance imaging (MRI) and brain histology in a mouse model.

Methods:

SAH was induced by endovascular perforation in adult mice. First, adult male wild-type (WT) mice underwent MRI T2 and T2* 4 hours after an endovascular perforation or a sham operation and were euthanized to assess brain histology. Second, male and female adult lipocalin-2 knockout (LCN2 KO) mice had SAH. All animals underwent MRI at 4 hours and the brains were harvested for brain histology.

Results:

T2* hypointensity vessels were observed in the brain 4 hours after SAH in male WT mice. The numbers of T2* positive vessels were significantly higher in SAH brains than in sham-operated mice. Brain histology showed thrombosis and erythrocyte plugs in the T2* positive cerebral vessels which may be venules. The number of T2* positive vessels correlated with SAH grade and the presence of T2 lesions. Brain thrombosis was also accompanied by albumin leakage and neuronal injury. LCN2 deficient male mice had lower numbers of T2* positive vessels after SAH compared with WT male mice.

Conclusions:

SAH causes ultra-early brain vessel thrombosis that can be detected by T2* gradient-echo sequence at 4 hours after SAH. LCN2 deficiency decreased the number of T2* positive vessels.

Graphical Abstract

Subarachnoid hemorrhage (SAH) causes ultra-early brain vessel thrombosis that can be detected by T2* gradient-echo sequence at 4 hours after SAH. Formation of cerebral vessel thrombosis results in acute blood-brain barrier (BBB) leakage and neuronal injury. Lipocalin-2 knockout (LCN2 KO) decreases the number of T2* positive vessels.

Introduction

Subarachnoid hemorrhage (SAH) is a high mortality and morbidity form of stroke, which accounts for 30% of death in patients and results in long-term cognitive deficits in 50% of survivors.^{1, 2} Pathological events, such as elevated intracranial pressure, decreased cerebral blood flow (CBF) and impaired CBF autoregulation, are immediately induced by SAH and these events initiate inflammation and oxidative stress which result in blood-brain barrier (BBB) disruption, brain edema, as well as neuronal death.^{3, 4} Early diagnosis and treatment have an important role for potential reduction of the mortality after SAH.

SAH has early effects on the microvasculature. Endothelial cell damage, basal lamina degradation, platelet aggregation, coagulation cascade activation and thrombo-inflammation happen within minutes after SAH which directly target microvessels.⁵ These changes lead to BBB disruption, microvessel spasm and microvascular thrombosis. Thrombosis occurs after SAH in patients and in experiment animal models,^{6, 7} little is known about thrombosis in venules after SAH, but there is evidence that thrombi form in pial veins.^{7, 8}

Magnetic resonance imaging (MRI) provides non-invasive structural, physiological and functional imaging data of the whole brain. We have found that white matter injury induced by SAH can be detected by MRI as T2-hypertensity at 4h and 24h time-point.^{9, 10} However, changes in T2* gradient-echo sequences have not been widely explored, especially in its hyperacute phase after SAH. Thrombotic veins were detected on T2* gradient-echo sequences as a loss of signal resulting from the susceptibility effects of deoxyhemoglobin within the blood clots,^{11, 12} this occurs as red thrombi at the site of venous occlusion. Because of thrombotic changes of a small, cerebral cortical vein are often subtle, and the intravascular clot may not be distinguishable from the normal blood flow signal, T2* gradient-echo sequences may be better suited since their considerably higher sensitivity to susceptibility differences. We applied T2* gradient-echo sequences to examine vascular changes after SAH and histology to examine underlying mechanisms.

Lipocalin-2 (LCN2) is a member of the highly heterogeneous secretory protein family of lipocalins. We have previously reported a role of LCN2 in white matter injury at 4 hour and 24 hour after SAH.^{9, 10} LCN2 had been reported that it is implicated in microvessel damage, BBB disruption and neuroinflammation after ischemia.¹³ Whether or not LCN2 playing a role in thrombosis after SAH is still unknown.

The current study examined the value of T2* gradient-echo sequences in diagnosing thrombosis after SAH. The effects LCN2 on SAH-induced ultra-early thrombosis were also examined.

Materials and methods

The authors declare that all supporting data are available within the article.

Animal preparation and endovascular perforation model

All animal protocols were approved by the University of Michigan Committee on the Use and Care of Animals. The University of Michigan has an Animal Welfare Assurance on file with the Office for Protection from Research Risks and is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. The studies followed the Guide for The Care and Use of Laboratory Animals (National Research Council) and comply with the ARRIVE guidelines for reporting in vivo experiments.

Mice were housed under standard 12:12 light-dark conditions and allowed free water and food. A total of 73 adult mice, including 51 male wild-type (WT) C57BL/6 mice (3 months old; Charles River Laboratories) and 22 adult (3 months old) male/female LCN2 knockout (LCN2 KO) mice (University of Michigan Breeding Core) were used in this study. Two male LCN2 KO mice died after SAH and were excluded from this study.

SAH was induced by endovascular perforation technique as previously described.¹⁰ Briefly, mice were anesthetized with 5% isoflurane and a controlled heating pad was used to keep core body temperature at 37.5°C. After induction of anesthesia, isoflurane was maintained at 1.5%. Under a microscope, a middle skin incision was made to expose the left common carotid artery, external carotid artery and internal carotid artery. Following sectioning of the left external carotid artery, a 5–0 monofilament suture was inserted into the left internal carotid artery until resistance was felt and carefully pushed further to perforate the artery. The suture was then withdrawn to induce SAH. Sham control mice underwent the same procedure without perforation.

Experimental groups

There were two parts to this study. First, 51 adult male WT mice randomly underwent an endovascular perforation (n=34) or a sham operation (n=17) at ratio of 2:1(https://www.randomizer.org). All animals underwent MRI after 4 hours and were then euthanized for brain histology and immunohistochemistry. Second, 14 adult male LCN2 KO and 8 adult female LCN2 KO mice underwent an endovascular perforation before undergoing MRI at 4 hours.

Magnetic resonance imaging and T2* positive vessel measurements

MRI was performed at 4 hours after SAH using a 9.4-T Varian MR scanner (Varian Inc) with acquisition of T2 fast spin-echo and T2* gradient-echo sequences using a field of view of 20 × 20 mm, matrix of 256 × 256 mm, and 25 coronal slices (0.5 mm thick)¹⁴. Mice were anesthetized with 1.5% isoflurane throughout the MRI examination. For each T2* gradient-echo image, three regions of interest (ROI: cortex, hippocampus, basal ganglia) were manually segmented on coronal sections (supplementary materials, Figure I). For each ROI, the number of hypointense vessels (T2* positive) was counted and summed across sections. All image analysis was performed using Image J software by a blinded observer.

SAH grades and MRI grades

The extent of SAH was assessed using a modified grading system as previously described.¹⁰ The basal brain including brainstem was divided into 6 segments. Each segment was assigned a grade from 0 to 3, depending on the amount of blood. The minimum SAH grade is 0 and maximum grade is 18. The MRI grading of SAH was also used as previously described¹⁴. Animals were categorized into the following five grades by T2* imaging; grade 0 indicates no visible SAH or intraventricular hemorrhage (IVH); grade 1, minimal/localized SAH with no IVH; grade 2, minimal/localized SAH with IVH; grade 3, thick/diffuse SAH (hematoma exceeding 0.5mm thickness is visible in more than 2 slices of T2* image) with no IVH; and grade 4, thick/diffuse SAH with IVH.

Brain histology and hematoxylin & eosin (H&E) staining

Mice were anesthetized (pentobarbital, 60 mg/kg i.p.) and perfused transcardially with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were fixed in 4% paraformaldehyde for 6 hours and then immersed in 30% sucrose for dehydration for 3–4 days at 4°C. Brains were then sectioned on a cryostat (18-μm-thick slices). H&E staining was performed following a standard protocol and sections were observed by light microscopy.

Immunohistochemistry

Immunohistochemistry was performed using the avidin-biotin complex technique as previously described.¹⁵ Polyclonal rabbit anti-alpha smooth muscle actin (αSMA) antibody (1:200; ab5694; Abcam), polyclonal goat anti-mouse albumin antibody (1:1000; A90–134P; Bethyl Laboratories), monoclonal rat anti-Ly76 antibody (1:200, ab91113; Abcam), and polyclonal rabbit anti-NeuN antibody (1:500; ab104225; Abcam) were used. Goat anti-rabbit IgG (1:500; Cat# 31820, Invitrogen) and goat anti-rat IgG (1:500; BA-9400, Vector) were used as the secondary antibodies. Negative control procedures included omission of the primary antibody.

Statistical Analysis

Counting and measurements were performed by personnel blinded to the experimental groups. Quantitative data are presented as the mean ± SD. Comparisons were performed by Student t test or Welch’s t test when p<0.05 on a F test. A p value of <0.05 was considered to be statistically significant.

Results

In this hyperacute (4 hour) study, mortality rates were 0% (0/34), 14% (2/14) and 0% (0/8) after endovascular perforation in male WT, male LCN2 KO mice and female LCN2 KO mice respectively. No sham mice died (n=17 male WT). All dead mice were excluded from analyses for failing to accomplish the entire course of the experiment.

T2* positive vessels were observed in WT mice 4 hours after endovascular perforation (Figure 1A). Although some T2* positive vessels were found in sham-operated WT mice, the total number of T2* positive vessels was significantly greater after SAH (23.9 ± 20.4 vessels/brain) compared to sham (6.5 ± 3.1 vessels/brain; p<0.01; Figure 1B). The number of T2* positive vessels was determined twice. The intra-observer variability was 1.9 ± 1.8 vessels/brain (mean ± SD) and 0.7 ± 0.8 vessels/brain in the SAH group and the sham group, respectively. The mean differences corresponded to 1.2 and 4.4% of the average number of vessels in those groups. H&E staining showed that microvessels had thrombosis and were blocked by erythrocytes, and the perivascular space was widened (Figure 1C). These vessels had only a single layer of αSMA-positive smooth muscle, suggesting they were venules (Figure 1D).

Figure 1. Subarachnoid hemorrhage (SAH) induces the appearance vessels that are hypointense on T2* gradient-echo images and that are obstructed with erythrocytes on histology.

A, Representative coronal T2* gradient-echo images 4 hours after a SAH or a sham operation. Numerous hypointense vessels were observed after SAH in the cortex but also in the anterior choroidal vein and its branches. B, The total number of T2* positive vessels in sham and SAH groups. Values are mean ± SD, n=17 in sham group, n=34 in SAH group, #p<0.01 vs sham group. C, Representative H&E Staining shows obstructed microvessels in SAH group compared with sham group. Arrow heads indicate aggregation of erythrocytes in thrombotic microvessels. Note also the dilated perivascular space in the SAH animal. Low magnification scale bar=20 μm, High magnification scale bar =10 μm. D, The type of thrombotic microvessels was examined using αSMA immunohistochemistry. A single layer of αSMA positive smooth muscle suggests they were venules. Low magnification scale bar=50 μm, High magnification scale bar =20 μm.

Hypointense cortical veins have been observed in rats under isoflurane anesthesia (dose 2%) using 7T T2*-gradient echo sequences by contributing to blood oxygenation level-dependent contrast¹⁶. The nature of the T2* positive was, therefore, examined further. H&E staining showed that T2* positive vessels in the cortex and basal ganglia in the sham group were of large diameter (>30 μm). No hypointense vessels were detected in the hippocampus (Figure 2A) though vessels with a diameter >30 μm were present. In contrast, H&E staining showed that T2* positive vessels in the SAH group have smaller diameter (<30 μm), but also have microthrombi and erythrocyte plugs (Figure 2A). Compared with sham group, the number of T2* positive vessels after SAH was significantly increased in cortex (18.9 ± 17.3 vs 4.5 ± 2.5 vessels in sham, p<0.01, Figure 2B), hippocampus (0.8 ± 1.1 vs 0 vessels in sham, p<0.01, Figure 2C) and basal ganglia (4.2 ± 3.6 vs 2.0 ± 1.6 vessels in sham, p<0.01, Figure 2D).

Figure 2. Thrombotic vessels can be detected by T2* gradient-echo sequence after SAH.

A, T2* images from sham-operated and SAH mice and corresponding H&E stained sections. In sham-operated mice, T2* positive vessels cortex and basal ganglion had large diameters (> 30 μm). No T2* positive vessels were observed in hippocampus in sham-operated mice, even when they had a diameter > 30 μm. In SAH mice T2* positive vessels in cortex, hippocampus and basal ganglion had microthrombi and microclots and diameters < 30 μm. The location of the areas was in relation to MRIs in the same animals. Low and high magnification scale bar = 100 and 20 μm, respectively. B-D, Numbers of T2* positive vessels in cortex, hippocampus and basal ganglion are significantly higher in SAH group compared to sham group. Values are mean ± SD, n=17 in sham group, n=34 in SAH group, #p<0.01 vs sham group.

To evaluate the relationship T2* positive vessels and the SAH severity and hemorrhage distribution, animals were divided into subgroups using either SAH grading or MRI grading systems. The total number of T2* positive vessels in mice with severe SAH, a SAH grade score of 10–18, was significantly higher (26.7 ± 21.9 vessels/brain) than in mice with low SAH grade scores of 1–9 (12.7 ±6.3 vessels/brain, p<0.01, Figure 3A). Comparing different regions of brain, the numbers of T2* positive vessels in cortex, hippocampus, and basal ganglia in high SAH grade mice (20.9 ±18.6, 0.9±1.2 and 4.9 ± 3.7 vessels, respectively) were significantly higher than that in low SAH grading mice (10.9 ± 6.9 vessels in cortex, p<0.05; 0.3 ± 0.5 vessels in hippocampus, p<0.05; 1.6 ± 1.6 vessels in basal ganglia, p<0.01; Figure 3A). These results indicated that the number of T2* positive vessels might correlate with SAH severity. In the cortex, the number of T2* positive vessels in mice with thick/diffuse SAH, MRI grade 3 and 4, was significantly higher (26.6 ± 20.1 vessels) compared to mice with minimal/localized SAH, MRI grades 0, 1 or 2 (14.4 ± 12.6, vessels, p<0.05, Figure 3B), indicating that T2* positive cortical vessels were associated with hemorrhage distribution after SAH. In addition, the number of T2* positive vessels in hippocampus in the mice with IVH, MRI grades 2 and 4, was significantly higher (1.8 ± 1.5 vessels) than those without IVH, MRI grades 0, 1 or 3 (0.4 ± 0.6 vessels, p<0.05, Figure 3B).

Figure 3. The number of T2* positive vessels after SAH increased with hemorrhage severity.

A. Total number of T2* positive vessels in mice of low and high SAH grade groups (SAH grading score 1–9 and 10–18, respectively). Values are mean ± SD, n=7 in low SAH grade group, n=27 in high SAH grade group, #p<0.01 vs low SAH grade group. The number of T2* positive vessels was also compared between low and high SAH grade groups in cortex, hippocampus and basal ganglia. Values are mean ± SD, n=7 in low SAH grade group, n=27 in high SAH grade group, *p<0.05 in cortex, *p<0.05 in hippocampus, and #p<0.01 in basal ganglia vs low SAH grade group. B. Number of T2* positive vessels in the cortex in mice with minimal/localized SAH (MRI grade 0, 1 or 2) or thick/diffuse SAH (MRI grade 3 or 4). Values are mean ± SD, n=20 in minimal/localized SAH group, n=14 in thick/diffuse SAH group, *p<0.05 vs minimal/localized SAH group. Number of T2* positive vessels in the hippocampus in mice without intraventricular hemorrhage (IVH, MRI grades 0, 1 or 3) or with IVH (MRI grade 2 or 4). Values are mean ± SD, n=25 in without IVH group, n=9 in with IVH group, *p<0.05 vs without IVH group.

To investigate the effects of thrombosis on BBB disruption and neuronal injury 4 hours after SAH, albumin leakage around the thrombotic vessels and NeuN positive cells were examined. Mice that underwent SAH but not a sham operation developed erythrocyte plugs in capillaries and other microvessels at 4 hours (Figure 4A). SAH also induced albumin leakage (BBB disruption) along the thrombotic microvessels (Figure 4B). We also found there were neuronal damage both in cortex and hippocampus at this hyperacute stage after SAH (Figure 4C, D).

Figure 4. Thrombotic vessels were associated with erythrocyte plugs, BBB disruption and neuron injury.

A, Representative immunohistochemistry of cortex in sham and SAH groups. In contrast to the sham group, erythrocyte plugs (Ly76 positive) obstruct capillaries and larger microvessels 4 hours after SAH. Low magnification scale bar=50 μm, high magnification scale bar =20 μm. B, Representative albumin immunohistochemistry in cortex with albumin leakage out of a thrombotic vessel (arrowheads) after SAH, but no leakage from a similar sized vessel group compared with sham group. Low magnification scale bar=50 μm, High magnification scale bar =20 μm. C, Representative NeuN immunohistochemistry of cortical neurons in SAH sham groups. Neuron (NeuN positive cells) in SAH group were reduced compared to the sham group. Low magnification scale bar=50 μm, High magnification scale bar =20 μm. D, Representative NeuN immunohistochemistry of hippocampal neurons in SAH and sham groups. Note the marked neuronal loss after SAH compared to the sham group. Low and high magnification scale bars =100 μm and 50 μm, respectively.

On T2 MRI, 35% (12/34) mice had a T2 hyperintensity (T2 lesion) in parenchyma after SAH, while 65% (22/34) had no T2 lesion. Whether the number of T2* positive vessels was related to the presence of T2 lesions was examined (Figure 5). The total number of T2* positive vessels in mice with a T2 lesion (39.0 ± 26.5 vessels/brain) was higher than tin animals without a T2 lesion (15.6 ±9.3 vessels/brain, p<0.05) in SAH group (Figure 5B). This suggests thrombosis may contribute to T2 lesion formation.

Figure 5. The number of T2* positive vessels was increased in mice with T2 lesions.

A, Examples of MRIs showing T2* positive vessels in mice with or without T2 lesion after SAH. T2 lesions were associated with higher numbers of T2* positive vessels. B, Quantification of the total number of T2* positive vessels in mice with and without T2 lesions. Without T2 lesion group n=22, with T2 lesion group n=12; *p<0.05 vs without T2 lesion group.

To examine whether LCN2 deficiency reduces the number of T2* positive vessels, WT and LCN2 KO male mice were examined 4 hours after SAH (Figure 6A). The SAH MRI grade was similar in male WT and LCN2 KO mice (1.9 ±1.3 and 2.5 ± 1.2, respectively; p>0.05) (Figure 6B). Compared with male SAH WT mice (23.9 ±20.4 vessels/brain), the total number of T2* positive vessels was significantly reduced in male LCN2 KO mice after SAH (15.3 ± 8.6 vessels/brain, p<0.05, one-tailed, Figure 6C).

Figure 6. The number of T2* positive vessels after SAH was less in male LCN2 deficient mice.

A, Examples of T2* positive vessels in WT and LCN2 KO mice. B, The SAH MRI grade was equivalent in the WT and LCN2 KO groups. n=34 in WT group, n=12 in LCN2 KO group, NS = not significant. C, Compared with WT group, the number of T2* positive venules significantly lower in LCN2 KO group after SAH. Values are mean ± SD, n=34 in WT group, n=12 in LCN2 KO group, **p<0.05 one-tailed vs WT group.

We also counted the total number of both sexes in LCN2 KO mice to examine sex differences. There were no significant differences in MRI grade between female LCN2 KO mice (3.3 ± 0.9) and male LCN2 KO mice (2.5 ± 1.2, p>0.05), or the total number of T2* positive vessels between female and male LCN2 KO mice (10.0 ±5.6 vs.15.3 ± 8.6 vessels/brain).

Discussion

The main finding in this study is that we discovered that T2* MRI detects cerebral vessel thrombosis by 4 hours after SAH. Secondly, the number of such vessels was increased with higher SAH grade and the presence of T2 lesions. Thirdly, LCN2 deficiency reduced such thrombosis after SAH.

Muroi et al. have described “markedly hypointense cortical veins” on T2* weighted images (T2*WI) in 7 T MRI after subarachnoid hemorrhage in mice.¹⁷ They suggested this was due to CBF reduction without pathological confirmation. In contrast, many articles have described that T2* gradient-echo sequence is the best method to detect cerebral vein thrombosis or dilated veins due to such thrombosis.^{11, 12} T2* gradient-echo sequence is more sensitive to paramagnetic effects than spin echo-based techniques. Paramagnetic compounds (such as deoxyhemoglobin, intracellular methemoglobin, and hemosiderin) can produce a nonuniform magnetic field and rapid dephasing of proton spins and loss of T2*-weighted signal due to its magnetic susceptibility effect. Alterations in blood flow and hemoglobin degradation products in thrombotic veins can produce signal changes on T2* gradient-echo sequence and is related to paramagnetic deoxyhemoglobin within trapped red blood cells in the thrombus. This property of paramagnetic molecules within the thrombi or venous obstruction due to thrombosis results in signal loss (hypointense). This effect is more apparent in acute stage of cerebral vein thrombosis because susceptibility effects are most pronounced in venous segments with more acute thrombi.¹¹ But the sensitivity of T2* gradient-echo sequence to diagnose cerebral thrombosis is still unknown. It is important to recognize that the hypointenstiy seen on T2* gradient-echo sequence does not always indicate intravascular thrombosis or dilation. Venous hypointensity was also described in acute ischemic stroke, that was attributed to blood oxygenation level dependent phenomenon due to a relative increase in intravenous deoxyhemoglobin.¹⁸ By using ultra-high field MRI (17.2 T), there were abundant hypointense veins viewed compared with high-field MRI viewed in cortex, hippocampus and basal ganglion in mouse brain without pathological basis.¹⁹ These results suggest that careful attention should be paid to differences in the magnetic field strength, with high field MRI (9.4 T) in our study being able to discriminate thrombotic vessels from normal. Further studies are needed to confirm the sensitivity of vessel hypointensity related to thrombosis and evaluate if increased numbers of hypointense vessels predicts worse functional outcome. More work is also required to determine whether T2* imaging preferentially identifies thrombotic veins/venules.

Microvascular thrombi were first identified in a SAH patient autopsy in 1983.²⁰ Since then, several autopsy studies have confirmed the presence of microvascular thrombi in SAH patients.²¹ In experimental SAH, microvascular thrombi were first described in cortex and cerebellum eight days after SAH.²² Sabri et al. and Vergouwen et al. described microvascular thrombi formation in cortex and hippocampus 24 hours after SAH.^{6, 23} Pisapia et al. described the time course of microvascular thrombi formation 24, 48, 72, and 96 hours after induction of SAH.²⁴ They observed microvascular thrombi in both ipsi- and contralateral hemispheres throughout at all time-points, but the severity peaked at 48 hours. Besides these fixed tissue studies with an earliest time-point of 24 hours after SAH, there were a few studies investigating pial vessels microthrombi using direct visualization by in-vivo video-microscopy within the first few hours after SAH in experimental animals.^{7, 25} Sun et al. found constricted pial arterioles and venules within 2 hours of SAH in rats. They also found agglutination of red blood cells and oscillations and cessations in blood flow, indicating formation of microthrombi.²⁵ Friedrich et al. suggested that more microarterial constriction in line with microthrombi, but there was no change in pial veins within 6 hours.⁷ Despite the direct observation of microthrombi in pial vessels, little is known about parenchymal vessels at the hyperacute stage of SAH, especially in veins/venules. Ishikawa et al. first observed thrombogenic responses in pial veins immediately after SAH in mice and they increased with time.⁸ Pisapia et al. confirmed the presence of microthrombi in arterioles, veins, and venules 24 hours after SAH.²⁴ Sun et al. showed an enlarged hypointense band on magnetic resonance venography that peaked at 3 hours and returned to normal value on day 2 after SAH, effects that were attributed to changes in cortical vein dilation.²⁶ Combined with the current study, this change may be associated with increased intravascular pressure and increased microvascular permeability due to vein thrombosis, leading to blood component extravasation. We observed enlarged extravascular space and albumin leakage out of thrombotic vessels that were probably veins. Those results combined with our recent findings on albumin leakage in white matter,⁹ indicate that BBB disrupted in thrombotic vessels and white matter 4 hours after SAH. Besides albumin leakage, the current study also found neuronal death in cortex and hippocampus at this hyperacute phase of SAH.

The current study found that the number of T2* positive micro-vessels in the brain was increased significantly at 4 hours after SAH. Most of these T2* positive vessels were thrombotic. However, a few T2* positive vessels were also detected in sham brain. Future studies should develop novel MRI protocols to differentiate normal vessels from thrombotic vessels. It is also unclear whether the thrombotic microvessels were venules or arterioles, which should be determined in future studies. These vessels had only a single layer of αSMA-positive smooth muscle, suggesting they might be venules. However, in a mouse model of SAH, Ishikawa et al. showed marked changes in arterioles during the first 60 minutes²⁷.

The present results suggest that thrombosis is a therapeutic target in SAH and the T2* imaging can be used to monitor such thrombosis. Such a therapeutic approach would run the risk of rebleeding but might be safer after clipping or embolization of aneurysms. Intraventricular fibrinolysis is currently being examined clinically for SAH (NCT03187405). T2* imaging may be useful for monitoring the efficacy of intravascular thrombolytic therapy for SAH and other cerebrovascular diseases.

Previous results indicate that LCN2 contributes to SAH-induced white matter injury and BBB disruption 4 hours after SAH.⁹ The present study provided evidence that LCN2 deficiency attenuates vessel thrombosis, which may result in reduced BBB albumin leakage and neuronal death. LCN2 deficiency also reduces neuroinflammation and oxidative stress at 24 hours after ICH as well as thrombin-induced brain edema.^{28, 29} The exact mechanisms by which LCN2 is involved in BBB disruption, white matter injury, and vascular thrombosis are still unclear. LCN2 is predominantly expressed in endothelial cells and its receptor is also detected in endothelial cells in mice after transient middle cerebral artery occlusion.³⁰ The LCN2 receptor is also expressed in human endothelial cells.³¹ Jin et al. also found that LCN2 deficiency attenuates neuroinflammation, vascular permeability and rescues down regulation of tight junction after ischemia.³⁰ Kim et al. recently discovered microvessel damage; BBB disruption and neuroinflammation were reduced in LCN2 deficiency mice after ischemia.¹³ They suggested that the hypoxia-inducible factor-1 alpha -LCN2- vascular endothelial growth factor A axis elicits vascular hyperpermeability. Neuroinflammation and injured endothelial cells participate in microvessel thrombus formation early after SAH.⁵ Therefore, LCN2 may play roles in vessel thrombosis after SAH through neuroinflammation. However, the causes of cerebral vessel thrombosis are still unknown. Whether thrombin formation following SAH has a role should be examined.

There are limitations to this study. This study focused on a single time point (4 hours). It will be important to determine how well T2* imaging can be used to monitor the full-time course of cerebral thrombosis after SAH. In addition, we have not definitively identified the vessels involved in the early thrombosis. Our results suggest that venules and veins are involved but this requires further investigation. In addition, in this proof-of-concept study, power analysis was not performed.

In conclusions, our findings suggest a new therapeutic target for the treatment of SAH. It may be necessary to perform anticoagulant therapy or thrombolysis as soon as possible after embolization or clipping of aneurysm.

Non-standard Abbreviations and Acronyms

αSMA

alpha smooth muscle actin

BBB

blood-brain barrier

CBF

cerebral blood flow

H & E

hematoxylin & eosin

KO

knockout

IVH

intraventricular hemorrhage

LCN2

lipocalin-2

MRI

magnetic resonance imaging

NeuN

neuronal nuclei

ROI

region of interest

SAH

subarachnoid hemorrhage

WT

wild-type