Lipocalin-2 released in response to cerebral ischaemia mediates reperfusion injury in mice

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin-2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose-dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke-reperfusion injury.


Regional cerebral blood flow (rCBF)
During focal cerebral ischemia, rCBF was continuously monitored by Laser Doppler Flowmetry (PF5001, Perimed) with a flexible 0.8-mm fiber optic extension probe (Probe 407, Perimed). The tip of the probe was affixed to the intact skull over the right cortex at 2 mm posterior to the bregma and 4 mm lateral to the midline. The rCBF was recorded 20 minutes before ischemia to obtain a steady-state baseline (100%) and continued until 15 minutes after the reperfusion. The percentage of the baseline rCBF (%) was calculated as the percentage relative to the baseline. The abrupt drop of Laser Doppler signal to ~20% of the baseline rCBF indicated the occlusion of MCA by the suture. Mice were excluded from further studies if sufficient occlusion (<30% of the baseline) and reperfusion (>80% of the baseline) was not achieved based on the Laser Doppler Flowmetry, if excessive bleeding occurred during surgery, or if hemorrhage was found in the brain slices or at the base of the circle of Willis during postmortem examination.

Neurological deficit scores and corner tests
Twenty-three hours after the initiation of ischemic stroke, mice were evaluated for neurological deficits using a four-tiered grading system [3,5]. Score 0 indicates no observed neurological deficit; score 1, inability to walk straight; score 2, circling toward the paretic side; score 3, falling on the paretic side; and score 4, loss of the righting reflex. For the corner test, mice were placed between two vertical boards attached at a 30° angle [6]. The number of right (ipsilateral) turns when the mouse reached the wedge of the corner was recorded in ten trials.

Collection of mouse serum
At different time points after ischemic stroke, mice were anesthetized with 5% isoflurane and euthanized by cervical dislocation. The blood was collected from the decapitated trunk and placed at room temperature for one hour. The blood was centrifuged at 2000 X g for 20 min at room temperature, and the supernatant was collected as blood serum for western blotting and ELISA [7].

Determination of infarct volume and brain swelling
Twenty-three hours after one hour of tMCAO, mice were anesthetized with 5% isoflurane and euthanized by cervical dislocation. The brain was removed and sliced into 1-mm-thick coronal sections using a brain matrix (Braintree Scientific) on ice. The brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) in 1X PBS at room temperature for 20 minutes and fixed in 10% formalin (Sigma-Aldrich) at 4 °C until imaging. Both sides of sections were photographed using a Leica EZ4HD stereomicroscope with integrated High Definition digital camera. The areas of cerebral infarction and of hemispheres were measured using the NIH ImageJ by an investigator blinded to the treatment conditions. The total infarct volumes were calculated using the following equations to consider the effect of edema: infarct area on one side of brain section = area of contralateral hemisphere -(area of ipsilateral hemisphere -area of infarct area) [8,9]. Total infarct volume = (front infarct area of a section + rear infarct area of the same section) / 2 × thickness of the section × total numbers of sections. Brain swelling (the amount of edema formation) = [(ipsilateral hemisphere volumecontralateral hemisphere volume) / contralateral hemisphere volume] × 100.

Primary neuronal culture and oxygen glucose deprivation (OGD)
Primary mixed neuronal-glial cultures were prepared from the cerebral cortex or hippocampus of postnatal day 1 (P1) to P3 mice as previously described [10][11][12]. Primary neuronal-glial cells were cultured in a humidified CO 2 incubator with 5% CO 2 and 95% air at 37 °C for 10-14 days in vitro (DIV [10][11][12][13][14]. To initiate OGD, culture media were changed into Neurobasal-A media without glucose (Invitrogen) containing B27 Supplement Minus AO (antioxidants), Glutamax, and sodium pyruvate [13]. The cultures were placed into a humidified Modular Incubator Chamber (Billups-Rothenberg) and flushed with 5% CO 2 and 95% N 2 for 5 min. The chamber was then sealed and incubated at 37 °C for one hour of OGD. To mimic reoxygenation, the culture was removed from the Modular Incubator Chamber and the media replaced with regular Neurobasal-A media with glucose and incubated with 5% CO 2 and 95% air at 37 °C for 23 hours.

ELISA
The level of LCN2 protein in mouse sera and brain homogenates was quantified following the manufacturer's protocol for mouse lipocalin-2/NGAL Quantikine ELISA kit (R&D).

MTT assays
The viability of cells after treatment with different concentrations of recombinant human LCN2 protein (R&D) was analyzed by MTT assays according to the protocols for the Vybrant® MTT Cell Proliferation Assay Kit (Molecular Probes). Briefly, cells were incubated with 1.2 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in a humidified CO 2 incubator at 37 °C for 4 hours. Cells were then lysed in SDS-HCl solution and incubated at 37 °C for 16 hours. The concentration of MTT formazan was measured by optical density (OD) at 570 nm. Cell viability was calculated by dividing the OD of treated group by the mean OD of untreated controls.

Visualization of cerebral vessels
The cerebral vessels were mapped as described [16]. Higgins Black Magic waterproof ink (200-250 µl; Sanford Corp.) was injected into the left ventricle using a 26-gauge needle, and the right atrium opened to release the effluent. Mice were decapitated and the heads fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 7 days. The brains were removed carefully from the skulls and imaged using a Leica EZ4HD stereomicroscope with integrated High Definition digital camera. The development of PcomA was graded on a qualitative scale of 0 to 3 [17]. Score 0 indicates no PcomA between anterior and posterior circulation; score 1 indicates PcomA in capillary phase; score 2 indicates small truncal PcomA; score 3 indicates truncal PcomA. To visualize the territory of MCA, we traced the peripheral branches of anterior cerebral artery (ACA) and MCA, and identified the points of anastomoses between ACA and MCA [3]. We connected the points of anastomoses to establish a "line of anastomoses". To assess the MCA territory, we measured the distances from the midline to the line of anastomoses at coronal planes 2, 4, and 6 mm from the frontal pole. Figure Legends Supplemental Figure S1. Assessment of cerebrovascular anatomy and determination of regional cerebral blood flow (rCBF). (A) Shown are representative images of the brains from Lcn2 +/+ and Lcn2 -/mice perfused with black ink. The major arteries in the circle of Willis (upper panels) and PcomA (indicated by arrowheads in lower panels) were identified. (B) Shown are the scores of PcomA plasticity in Lcn2 +/+ (n = 13) and Lcn2 -/mice (n = 13). (C) Shown are the dorsal images of the brains from Lcn2 +/+ and Lcn2 -/mice perfused with black ink. The points of anastomoses were circled and connected to form the line of anastomoses. (D) Distances from the line of anastomoses to the midline in Lcn2 +/+ (n = 3) and Lcn2 -/-(n = 3) mice were measured at coronal planes 2, 4, and 6 mm from the frontal pole. (E) Shown is a continuous tracing of Laser Doppler Flowmetry monitoring rCBF during one hour of tMCAO and reperfusion. Laser Doppler signal drops to ~12% of the baseline rCBF when the suture is advanced to the origin of MCA (arrow head), and returns back to the baseline when the suture is withdrawn (arrow). (F) The rCBF during tMCAO was measured continuously in Lcn2 +/+ (n = 8) and Lcn2 -/-(n = 8) mice. Steady-state rCBF before the MCAO were used as baseline (100%), and the subsequent changes after the onset of ischemia were shown as the percentage relative to the baseline. Time zero indicates the point of MCA occlusion. There were no significant differences in rCBF between Lcn2 +/+ and Lcn2 -/during tMCAO. Figure S2. Infarct size is similar in WT and LCN2 null mice after pMCAO.