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. Author manuscript; available in PMC: 2017 Apr 1.
Published in final edited form as: Stroke. 2016 Feb 11;47(4):1078–1084. doi: 10.1161/STROKEAHA.115.012153

Role of Lipocalin-2 in Thrombin-induced Brain Injury

Shanshan Mao 1, Guohua Xi 1, Richard F Keep 1, Ya Hua 1
PMCID: PMC4811682  NIHMSID: NIHMS755459  PMID: 26869387

Abstract

Background and Purpose

Thrombin and lipocalin-2 (LCN2) contribute to intracerebral hemorrhage-induced brain injury. Thrombin-induced brain damage is partially through a thrombin receptor, protease-activated receptor-1 (PAR-1). LCN2 is involved in cellular iron transport and neuroinflammation. The present study investigated the role of LCN2 in thrombin-induced brain injury.

Methods

There was three parts in this study. First, male adult C57BL/6 wild type (WT) or LCN2 knockout (LCN2 KO) mice had an intracaudate injection of thrombin (0.4U) or saline. Second, LCN2 KO mice had an injection of thrombin (0.4U) with recombinant mouse LCN2 protein (1µg) into the right caudate. Third, PAR-1 KO or WT mice had an intracaudate injection of thrombin or saline. All mice had T2-weighted magnetic resonance imaging and behavioral tests. Brains were used for histology, immunohistochemistry and Western blotting.

Results

Intracerebral thrombin injection caused LCN2 upregulation and brain injury in mice. Thrombin-induced brain swelling, blood-brain barrier disruption, neuronal death and neurologic deficits were markedly less in LCN2 KO mice (p<0.05) and were exacerbated by exogenous LCN2 co-injection. In addition, thrombin injection resulted in less LCN2 expression and brain injury in PAR-1 KO mice.

Conclusions

Thrombin upregulates LCN2 through PAR-1 activation and causes brain damage.

Keywords: blood–brain barrier disruption, brain swelling, lipocalin-2, neuronal death, protease-activated receptor-1, thrombin

Introduction

Thrombin, a blood-derived serine protease, is an essential component of the coagulation cascade. It is produced in the brain either immediately after intracerebral hemorrhage (ICH) or after the blood-brain barrier (BBB) disruption. Thrombin has a key role in ICH-induced brain injury1, 2. A family of protease-activated receptors (PAR) has been identified, of which PAR-1, PAR-3, and PAR-4 are activated by thrombin3. PAR-1 is the main thrombin receptor subtype. It is linked to many intracellular signaling pathways and is involved in brain injury after hemorrhagic and ischemic stroke4, 5.

Lipocalin-2 (LCN2), a siderophore-binding protein, is involved in cellular iron transport6. It is an acute phase protein that is up-regulated in a variety of central nervous system injuries7–11. Our recent studies have demonstrated that brain LCN2 is upregulated after ICH and that ICH-induced brain injury is much less in LCN2 knockout (KO) mice12, 13. Thrombin and iron are two major factors causing brain damage after ICH1. Our data have shown that LCN2 has a role in iron-mediated brain damage12. It is still unclear whether LCN2 has a role in thrombin-induced brain damage.

In this study, we investigated the role of LCN2 in thrombin-induced brain edema formation, BBB disruption, neurologic deficits, neuroinflammation and neuronal death. We also investigated whether exogenous LCN2 protein can aggravate thrombin-induced brain injury in LCN2 KO mice. In addition, the role of PAR-1 on thrombin-induced LCN2 upregulation and brain injury was also examined.

Materials and Methods

Animal Preparation and Intracerebral Infusion

Animal use protocols were approved by the University of Michigan Committee on the Use and Care of Animals. A total of 92 male mice at age of 2–3 months were used in the study. One wild type mouse (the 3-day control group) died during initial anesthesia and it was excluded from this study. LCN2 KO and PAR-1 KO mice were from University of Michigan Breeding Core, and C57BL/6 wild-type mice were from Charles River Laboratories (Roanoke, IL, USA). Mice were anesthetized with ketamine (90 mg/kg, intraperitoneally; Abbott Laboratories, Chicago, IL, USA) and xylazine (5 mg/kg, i.p.; Lloyd Laboratories, Shenandoah, IA, USA). Body temperature was maintained at 37.5°C by a feedback-controlled heating pad.

Mice were positioned in a stereotaxic frame (Model 500, Kopf Instruments, Tujunga, CA, USA) and a cranial burr hole (1 mm) was drilled near the right coronal suture 2.5 mm lateral to the midline. A 26-gauge needle was inserted stereotaxically into the right basal ganglia (coordinates: 0.2 mm anterior, 3.5 mm ventral, and 2.5 mm lateral to the bregma). Rat thrombin (Sigma) in 10µl saline or thrombin (0.4 U) with or without recombinant mouse LCN2 protein (1µg, R&D System, Minneapolis, MN, USA) was infused at 2µL/min by a microinfusion pump. Control animals had a 10µL saline injection. After injection, the needle remained in position for 10 minutes to prevent reflux and then it was gently removed. The burr hole was filled with bone wax, and the skin incision was sutured closed.

Experimental Groups

This study included three parts. We used selection of odd or even numbers to randomize animals. In the first part, WT or LCN2 KO mice had 0.4 U of thrombin or 10µl saline injection (n=7 per group and per time point). In the second part, LCN2 KO mice (n=7 per group) had an intracerebral injection of thrombin in saline with mice LCN2 protein (1µg) or vehicle. In our preliminary study, intracaudate injection of 1µg LCN2 into LCN2 knockout mice did not cause changes in T2 MRI. In the third part, WT or PAR-1 KO mice received an intracerebral injection of thrombin (0.4U) in saline or saline alone (n=7 per group). Based on our prior study on the variance in ICH-induced brain swelling in mice14, we estimated that an n=7 per experimental group would be able to detect a 50% reduction in brain swelling with greater than 80% power. All mice were euthanized at 24 hours or 72 hours after T2-weighted magnetic resonance imaging (MRI) and behavioral testing. Brains were used for histology, immunohistochemistry and Western blotting.

Magnetic Resonance Imaging and Brain Swelling Measurement

Imaging was carried out in a 7.0-T Varian MR scanner (183-mm horizontal bore; Varian, Palo Alto, CA) at the University of Michigan12. Mice were anesthetized with 2% isoflurane/air mixture throughout MRI examination. Mice had a T2 fast spin-echo sequence (TR/TE=4000/60 msec) with twenty-five slices of 0.5-mm thickness. The images were preserved as 256 × 256 pixel pictures for brain swelling calculation in NIH image J. Brain swelling calculation was based upon seven every other sections whose center was the anterior commissure layer. The value was: volume of (ipsilateral hemisphere- contralateral hemisphere)/volume of contralateral hemisphere×100 %12, 14.

Immunohistochemistry

Immunohistochemistry and immunofluorescence double staining were performed as described previously15. For immunohistochemistry, the primary antibodies were goat anti-LCN2 IgG (R&D System; 1:200 dilution), goat anti-albumin IgG (Bathyl Laboratories, Inc., Montgomery, TX, USA; 1:4000 dilution), rabbit anti-DARPP-32 IgG (Cell Signaling Technology, Danvers, MA, USA, ; 1:500 dilution), and rabbit anti-myeloperoxidase (MPO) IgG (Abcam; 1:200 dilution).

For immunofluorescence double labeling, the primary antibodies were polyclonal rabbit anti-glial fibrillary acidic protein (GFAP) IgG (Millipore, Billerica, MA, USA; 1:400 dilution), polyclonal rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1) IgG (Wako, Richmond, VA, USA; 1:500 dilution) and polyclonal rabbit antineuronal-specific nuclear protein (NeuN) IgG (Abcam; 1:500 dilution), as well as those for LCN2. The secondary antibodies were Alexa Fluro 488-conjugated donkey anti-rabbit mAb and Alexa Fluro 594-conjugated donkey anti-goat mAb (Invitrogen, Grand Island, NY, USA; both at a 1:500 dilution). The double labeling was analyzed using a fluorescence microscope (Olympus, BX51).

Cell counting

Cell counting was performed on brain coronal sections. Three high-power images (×40 magnification) were taken in different area of basal ganglia using a digital camera. The counting of positive cells was performed by a blinded observer. All measurements were repeated three times and the mean value was used.

Western Blotting Analysis

Western blot analysis was performed as previously described15. Briefly, mice were perfused with 0.1mmol/L phosphate-buffered saline (pH 7.4) after euthanasia and the ipsi- and contralateral basal ganglia were sampled. Protein concentration was determined by Bio-Rad protein assay kit (Hercules, CA, USA), and 50µg protein of each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a hybond-C pure nitrocellulose membrane (Amersham, Pittsburgh, PA, USA). Membranes were probed with the following primary antibodies: polyclonal goat anti-LCN2 IgG (R&D System; 1:200 dilution), goat anti-albumin IgG (Bathyl Laboratories; 1:10,000 dilution), and rabbit anti-DARPP-32 IgG (Cell Signaling Technology; 1:10,000 dilution). The secondary antibodies were rabbit anti-goat IgG and goat anti-rabbit IgG (1:2,000 and 1:2,500 dilution; Bio-Rad). Antigen-antibody complexes were visualized with the ECL chemiluminescence system (Amersham) and exposed to Kodak X-OMAT film. The relative densities of bands were analyzed with NIH Image J.

Behavioral Tests

Forelimb use asymmetry and corner turn tests were used for behavioral evaluation as previous described16–18. Behavioral scores were evaluated by a blinded investigator.

Statistical Analysis

All the data in this study are presented as mean±S.D. Data were analyzed by Student’s t test or ANOVA test. Differences were considered significant at p<0.05.

Results

Thrombin-Induced Brain LCN2 Upregulation

Numerous LCN2-positive cells were found in the ipsilateral basal ganglia 24 hours after thrombin injection in WT mice. There were few LCN2-positive cells in the contralateral basal ganglia after thrombin injection, as well as in the ipsilateral basal ganglia after saline injection (Fig.1A). LCN2 protein levels in the ipsilateral basal ganglia were significantly higher after thrombin injection than in the contralateral basal ganglia (p<0.01) or in the ipsilateral basal ganglia with saline injection (0.76±0.21 vs. 0.24±0.05, p<0.01) (Fig.1B). LCN2 positive cells co-localized with GFAP (astrocyte marker) positive cells, but not with Iba-1 (microglia marker) or NeuN (neuronal marker) positive cells (Fig.1C).

Figure 1.

Figure 1

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Wild-type (WT) mice received an intracerebral injection of thrombin or saline. At 24 hours, the brains were used to examine: (A) Lipocalin-2 (LCN2) immunoreactivity in the ipsi- and contralateral basal ganglia. Scale bar=50µm; (B) LCN2 protein levels in the ipsi- and contra lateral basal ganglia (Western blot). Values (ratio to β-actin) are means±S.D.; n=4 per group, #p<0.01 vs. other groups; (C) Double labeling of LCN2 with GFAP (astrocyte marker), Iba-1 (microglia marker), and NeuN (neuronal marker), in the ipsilateral basal ganglia after thrombin injection. Note the co-localization of LCN2 with the astrocyte marker. Scale bar=20µm.

Thrombin Caused Less Brain Injuries in Lipocalin-2 KO Mice

Brain swelling was measured in T2-weighted MRI coronal sections 1 day or 3 days after thrombin injection. Thrombin caused marked brain swelling in WT mice (p<0.01, Fig.2A) but was much less in LCN2 KO mice (day-1: 3.2±0.6% vs. 10.1±1.4 %, p<0.01; day-3: 2.9±0.7% vs. 9.8±1.2%, p<0.01, Fig.2A).

Figure 2.

Figure 2

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Wild-type (WT) and lipocalin-2 knockout (LCN2 KO) mice received an intracerebral injection of thrombin or saline. At 24 hours or 72 hours, brain injury was assessed by: (A) T2-weighted MRI. Brain swelling after thrombin or saline injection was calculated as ((ipsilateral–contralateral hemisphere)/contralateral hemisphere))×100%. Values are means±S.D.; n=7 per group, #p<0.01 vs. other groups. (B) Forelimb use asymmetry and (C) corner turn test scores. Values are means±S.D.; n=7 per group, #p<0.01 vs. other groups.

Thrombin injection but not saline injection induced significant neurologic deficits (p<0.01, Fig. 2B, 2C). The neurologic deficits induced by thrombin were less in LCN2 KO mice as measured by forelimb use asymmetry test (day-1: 34±9% vs. 52±11% in WT mice, p<0.01; day-3: 29±9% vs. 49±8% in WT mice, p<0.01, Fig.2B) and corner turn test (day-1: 69±11% vs. 98±4% in WT mice, p<0.01; day-3: 63±8% vs. 94±6% in WT mice, p<0.01, Fig.2C).

Intracaudate injection of thrombin caused severe BBB disruption at day-1 and day-3 as indicated by albumin immunohistochemistry and Western blot of ipsilateral basal ganglia (day-1: 6033±1146 vs. 66±20 pixels in saline group, p<0.01, Fig.3A; day-3: Suppl Fig I). Thrombin-induced albumin leakage was significantly less in LCN2 KO mice (p<0.01, Fig.3A and Suppl Fig I).

Figure 3.

Figure 3

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Wild-type (WT) and lipocalin-2 knockout (LCN2 KO) mice received an intracerebral injection of thrombin or saline. At 24 hours, the brains were used to assess: (A) Albumin immunoreactivity and protein levels in the ipsilateral basal ganglia. Scale bar=1mm; values (pixels) are means±S.D.; n=4 per group, #p<0.01 vs. other groups. (B) Myeloperoxidase (MPO) immunoreactivity in the ipsilateral basal ganglia. Scale bar=20µm; values are means±S.D.; n=3 per group, #p<0.01 vs. other groups. DARPP-32 immunoreactivity (C) and protein levels (D) in the ipsilateral basal ganglia. Note reduced DARPP-32 immunoreactivity on the right side of the section (ipsilateral) after thrombin injection in the WT mouse. Scale bar=1mm; values (ratio to β-actin) are means±S.D.; n=4 per group, #p<0.01 vs. saline group, *p<0.05 vs. LCN2 KO group.

In addition, thrombin-induced neutrophil infiltration and caudate neuronal death were much less in LCN2 KO mice at both day-1 and day-3 (Fig.3B–D and Suppl Figs II & III). Myeloperoxidase (MPO) and DARPP-32 staining were used to detect neutrophils and viable medium-sized spiny neurons, respectively. DARPP-32 protein levels were also measured by Western blots.

Effects of Exogenous LCN2 on Thrombin-induced Brain Injury in LCN2 KO Mice

To further confirm the effect of LCN2 on thrombin-induced brain injury, we evaluated whether co-injection of recombinant mouse LCN2 (1µg) in LCN2 KO mice would block the attenuation of thrombin-induced brain injury found in those mice. Co-injection of LCN2 with thrombin resulted in more severe brain swelling, BBB leakage, inflammation, neuronal death and neurological deficits compared to thrombin plus vehicle (Fig.4).

Figure 4.

Figure 4

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LCN2 KO mice received an intracerebral injection of thrombin + LCN2 or thrombin + vehicle. Brain injury at 24 hours was assessed by: (A) T2-weighted magnetic resonance imaging (MRI). Brain swelling was calculated as ((ipsilateral–contralateral hemisphere)/contralateral hemisphere) ×100%. Values are means ± S.D.; n=7 per group, #p<0.01 vs. vehicle-treated group. Blood-brain barrier permeability was examined using albumin immunoreactivity and albumin protein levels in the ipsilateral basal ganglia. Scale bar=1mm; values (pixels) are means±S.D.; n=4 per group, #p<0.01 vs. vehicle-treated group. (B) Forelimb use asymmetry and corner turn tests. Values are means±S.D.; n=7 per group, #p<0.01 vs. vehicle-treated group. (C) Myeloperoxidase (MPO) immunoreactivity in the ipsilateral basal ganglia. Scale bar= 20µm; values are means±S.D.; n=3 per group, #p<0.01 vs. vehicle-treated group. (D) DARPP-32 immunoreactivity and protein levels in the ipsilateral basal ganglia. Scale bar=1mm; values (ratio to β-actin) are means ± S.D.; n=4 per group, *p<0.05 vs. vehicle-treated group.

Thrombin-Induced LCN2 Upregulation and Brain Injury via PAR-1

To examine whether thrombin upregulates brain LCN2 via the PAR-1 receptor, PAR-1 KO mice were used. Thrombin injection resulted in fewer LCN2 positive cells in PAR-1 KO than in WT mice at 24 hours (Fig.5A). LCN2 protein levels in the ipsilateral basal ganglia of PAR-1 KO mice were significantly lower after thrombin injection (0.43±0.10 vs. 0.75±0.06 in WT mice, p<0.01, Fig.5A). Thrombin-induced brain swelling, BBB disruption, neutrophil infiltration, neuronal death and neurological deficits were all less severe in PAR-1 KO mice compared to WT mice (Figs.5 & 6).

Figure 5.

Figure 5

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Wild-type (WT) and PAR-1 knockout (PAR-1 KO) mice received an intracerebral injection of thrombin or saline. At 24 hours, the brains were used to examine: (A) Lipocalin-2 (LCN2) immunoreactivity and protein levels in the ipsilateral basal ganglia. Scale bar=50µm; values (ratio to β-actin) are means±S.D.; n=4 per group, #p<0.01 vs. other groups. (B) Brain swelling using T2-weighted magnetic resonance imaging (MRI). Brain swelling was calculated as ((ipsilateral-contralateral hemisphere)/contralateral hemisphere) ×100%. Values are means ± S.D.; n=7 per group, #p<0.01 vs. other groups. (C) Forelimb use asymmetry and (D) corner turn preference. Values are means±S.D.; n=7 per group, #p<0.01 vs. other groups.

Figure 6.

Figure 6

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Wild-type (WT) and PAR-1 knockout (PAR-1 KO) mice received an intracerebral injection of thrombin or saline. At 24 hours, the brains were used to assess: (A) Albumin immunoreactivity and protein levels in the ipsilateral basal ganglia. Scale bar=1mm; values (pixels) are means ± S.D.; n=4 per group, #p<0.01 vs. other groups. (B) Myeloperoxidase (MPO) immunoreactivity in the ipsilateral basal ganglia. Scale bar= 20µm; values are means±S.D.; n=3 per group, #p<0.01 versus other groups. DARPP-32 immunoreactivity (C) and protein levels (D) in the ipsilateral basal ganglia. Scale bar=1mm; values (ratio to β-actin) are means±S.D.; n=4 for each group, #p<0.01 vs. saline group, *p<0.05 versus PAR-1 KO group.

Discussion

The major findings of the present study are as follows: 1) brain LCN2 levels were upregulated after intracerebral injection of thrombin; 2) LCN2 deficiency resulted in less thrombin-induced injury such as brain swelling, BBB disruption and neuronal death; 3) exogenous recombinant mouse LCN2 exacerbated thrombin-induced brain injury in LCN2 KO mice; and 4) thrombin induced less LCN2 upregulation and brain injury in PAR-1 KO mice.

Thrombin has a key role in ICH-induced brain injury1, 2. The concentration of prothrombin in plasma is high (1–5 µM) and 1-mL of whole blood can produce about 260 to 360 units of thrombin. Thus, a 30-µl clot could be expected to produce up to about 10 units of thrombin. In preliminary studies, both 0.5 and 1 unit thrombin caused high mortality in mice. Therefore, 0.4 unit thrombin was used for this study.

The current study found a marked increase in LCN2 levels in the ipsilateral basal ganglia after intracerebral thrombin injection. Most of the LCN2-positive cells were astrocytes. This finding is consistent with previous reports9, 11, 19, 20, although neurons, microglia, and endothelial cells can also express LCN212, 21. Our data also found that thrombin results in a significant neutrophil infiltration which may also contribute to increased brain LCN2 levels (LCN2 is also known as neutrophil gelatinase-associated lipocalin). The cause and effect of LCN2 upregulation and neutrophil infiltration need to be studied further.

The biological actions of thrombin are both receptor- and non-receptor mediated, e.g. PAR-1 activation and fibrinogen cleavage. In the current study, thrombin-induced LCN2 upregulation was reduced but not completely prevented in PAR-1 KO mice. This indicates that PAR-1 activation is an important regulator of LCN2 although thrombin may also act via other mechanisms, e.g. PAR-3 and -4 or a non-receptor mediated mechanism. Two factors that can upregulate LCN2 are the cytokines interleukin-1β (IL-1β)22 and tumor necrosis factor-α (TNF-α)23. Intracerebral injection of thrombin increases IL-1β levels, an effect abolished in PAR-1 KO mice24, and increases brain TNF-α levels25. This suggests that, at least in part, thrombin and PAR-1 regulation of LCN2 may be via pro-inflammatory mediators.

We have previously demonstrated that LCN2 contributes to brain injury after ICH12 and that thrombin is an important mediator of ICH-induced injury1, 2. To examine whether LCN2 is involved in thrombin-mediated brain injury, LCN2 KO mice were used. Thrombin formed after brain hemorrhages causes brain edema and BBB disruption26, 27. The current study found that thrombin-induced edema (swelling), BBB leakage and associated neutrophil infiltration were all markedly reduced in LCN2 KO mice. The precise mechanisms of thrombin-induced BBB disruption are still unclear, studies have shown that LCN2 can enhance iron overload in cells28 and preserve MMP-9 activity29.

Intracerebral injection of thrombin also induces significant neurologic deficits and neuronal death16. Thrombin-induced neurological deficits were reduced in LCN2 KO mice. In the current study, dopamine-and cAMP-regulated phosphoprotein, Mr 32kDa (DARPP-32), a cytosolic protein highly expressed in medium-sized spiny neurons of the striatum30, 31, was used to assess neuronal death. DARPP-32 is a reliable marker of neuronal injury in basal ganglia32. The loss of DARPP-32 after thrombin injection was also reduced in LCN2 KO mice. Emerging evidence from other brain diseases suggest that LCN2 may contribute to neuronal death. For example, it may contribute to neuroinflammation and neurogeneration in Alzheimer Disease23, 33.

Further confirmation of a detrimental effect of LCN2 came from experiments where exogenous LCN2 was administered by intracerebral injection to LCN2 KO mice. That caused enhanced thrombin-induced injury as assessed by brain swelling, BBB disruption, neuronal cell death and behavioral deficits. The role of LCN2 in the brain appears to be multi-faceted. It may increase brain injury by pro-inflammatory effects and by altering cell iron-handling34.

PAR-1 is involved in thrombin-induced brain damage after ICH35. In this study, thrombin-induced LCN2 upregulation was reduced in PAR-1 KO mice and those mice also had less thrombin-induced brain swelling, BBB leakage, neuronal death and neurologic deficits. Indeed, it is noticeable that the effects of the PAR-1 and the LCN2 KO on thrombin-induced brain injury are very similar in magnitude. This suggests that LCN2 upregulation may be a major mechanism by which PAR-1 activation causes brain injury, but that hypothesis requires further validation.

As well as thrombin, iron contributes to brain injury after ICH1. Our previous study showed that LCN2 was up-regulated in a rat ICH model and that treatment of iron chelator, deferoxamine, attenuated ICH-induced LCN2 up-regulation13. Our data also showed that intracerebral injection of iron causes less brain swelling and BBB leakage in LCN2 KO mice12. These results indicate that LCN2 has a role in iron- as well as thrombin-mediated brain injury after ICH and suggests that targeting LCN2 may inhibit multiple injury pathways. It should also be noted that thrombin can enhance iron toxicity36. Understanding the different roles of LCN2 is important in determining how to manipulate it therapeutically.

In conclusion, thrombin upregulates LCN2 through the thrombin receptor, PAR-1, and at least part of thrombin-mediated brain injury is via LCN2. Accumulating evidence indicates that thrombin is involved in brain injury following ischemic and hemorrhagic stroke, and LCN2 may be a new therapeutic target for stroke patients.

Supplementary Material

Supplementary Materials

Acknowledgments

Sources of Funding: This study was supported by grants NS-084049, NS-091545, NS-090925, NS-073595, and NS-079157 from the National Institutes of Health (NIH).

Footnotes

Potential Conflicts of Interest: We declare that we have no conflict of interest.

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Supplementary Materials

Supplementary Materials
NIHMS755459-supplement-Supplementary_Materials.pdf (3.6MB, pdf)

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