Key role of 15-LO/15-HETE in angiogenesis and functional recovery in later stages of post-stroke mice

This study sought to clarify the effects of 15-lipoxygenase/15-hydroxyeicosatetraenoic acid in angiogenesis and neurological functional recovery after cerebral ischaemic stroke in mice. In vivo, we performed behavioural tests to determine functional recovery after stroke. Double immunofluorescence staining of CD31 and Ki67/PCNA was performed to evaluate the effects of 15-lipoxygenase/15-hydroxyeicosatetraenoic acid on angiogenesis in an MCAO mouse model. In vitro, we investigated the effects of 15-hydroxyeicosatetraenoic acid on BMVEC proliferation and migration. Our results show that MCAO upregulates 15-lipoxygenase expression in a time-dependent manner, especially in later stages of post-stroke. We confirmed that cerebral infarct area was reduced and neurological dysfunction was gradually attenuated after stroke, while 12/15-lipoxygenase knockout mice exhibited the opposite effects. Furthermore, immunofluorescence studies revealed 15-lipoxygenase increased the proliferation of mouse brain vascular endothelial cells in a time-dependent manner, while 12/15-lipoxygenase knockout blocked these effects. Moreover, 15-hydroxyeicosatetraenoic acid promoted proliferation and tube formation in BMVECs. These results demonstrate positive influence of 15-lipoxygenase/15-hydroxyeicosatetraenoic acid in angiogenesis and neuronal recovery after ischaemic stroke in mice. We also confirmed the PI3K/Akt signalling pathway was necessary for the effects of 15-hydroxyeicosatetraenoic acid in regulation of BMVEC proliferation and migration, which may potentially be a novel target for the recovery from ischaemic stroke.

global neurological function as previously described 1 . The scoring system was as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by tail; 2, circling to affected side; 3, unable to bear weight on affected side; and 4, no spontaneous locomotor activity or barrel rolling.

Infarction volume measurement
The infarct analysis cohort was subjected to stroke, mice were euthanized by decapitation at different time point after reperfusion. Briefly, brains were removed and cut into five 2-mm coronal sections and stained with 1.5% 2, 3, 5-triphenyltetrazolium chloride (TTC) for 8 min at 38 °C. Slices were formalin-fixed (4%), digitalized and infarct volumes were analysed (Sigma Scan Pro) as previously described 2 . The final infarct volumes are presented as a percentage (percentage of contralateral structures with correction for edema) ± SD, as previously described (n = 10/group) 2,3 .

Adhesive-tape removal test
The adhesive-tape (sticky-tape) removal test is a measure of somatosensory dysfunction after cerebral ischaemia in mice 4 . Adhesive-backed tape (30 x 40 mm) was used as tactile stimuli placed on the distal-radial region of the left wrist, and the mean time to remove the tape was recorded. Animals were trained for 5 days once a day prior to stroke, and the latency to remove adhesive tape was measured on days 0, 1, 7, 14 and 21 after stroke (n = 10/group).

Accelerated rotarod test
The rotarod test is used to assess motor coordination and balance alterations after ischaemic brain injury in the rodent 5 . The rotarod apparatus consists of a striated rod (diameter 3 cm) subdivided into 5 areas (width 5 cm) by disks 25 cm in diameter. Mice (n = 10/group) were conditioned to the accelerating rotarod (Ugo Basile, France) for 5 days before MCA occlusion. To this end, mice were first placed on the apparatus during 30 s with no rotation and thereafter for 2 min with a constant low speed (4 rpm). They were tested until they achieved a criterion of remaining on the rotating spindle for 1 min. This procedure was performed only the first day of training. After 10 min rest, each mouse then received a single baseline trial on the accelerating rotarod in which the spindle increased in speed from 4 to 40 rpm over a period of 6 min.
The test trial was performed at different times after MCAO in accordance with adhesive-tape removal test. The maximum duration the animals were able to walk on the rotarod before falling was measured (maximum value 6 min). Mice were tested over 3 daily trials in the accelerated condition (4-40 rpm). The daily mean value was taken for each mouse and used for statistical analysis.

Immunohistochemistry
Mice were euthanized by decapitation at different time point after reperfusion.
Then we removed the brains and immersed them in 4% paraformaldehyde for overnight fixation. Then the tissues were dehydrated, cleared and embedded in paraffin wax. The paraffin blocks were cut into 5 μm thick sections. For immunohistochemistry, sections were deparaffinized and rehydrated in graduated alcohol. Then they were placed in sodium citrate buffer (0.1 mol/L, pH 6.0) and heated for 2 min for antigen retrieval, and then the sections were incubated with anti-15-LO (1:100) antibodies. After overnight incubation, the sections were exposed to the secondary antibodies (1:200) for the IgGs.
Sections were visualized with 3, 3-diaminobenzidine and counterstained using haematoxylin. Brown and yellow colours indicated positive stains.

Measurement of 15-HETE level
To examine whether OGD promotes the generation of endogenous 15-HETE

Double immunofluorescence staining
After dissecting out brains from mice, the brain tissues were fixed in 4% paraformaldehyde for 6 h, transferred to 10%, 20%, 30% sucrose in 0.1 mol/L phosphate buffer (pH 7.4) for 12 h respectively in order to cryoprotection, and stored at 4°C. Brain tissue was frozen in Tissue-Tek OCT compound (Sakura

SiRNA design and transfection
To block the expression of 15-LO protein, BMVECs were transfected with the corresponding siRNAs, which were designed and synthesized by

Immunocytochemistry
BMVECs were cultured on coverslips, which were covered in 24-well culture plates with polylysine. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 3% normal bovine serum at 37°C for 30 min, and incubated with anti-15-LO primary antibody (rabbit, 1:50) and anti-CD31 primary antibody (rat, 1:100) at 4°C overnight. After washing three times with PBS, the cells were incubated with FITC-conjugated secondary antibody (1:100) for 2 h and DAPI for 10 min. in the dark. The images were recorded by digital photomicrography (Olympus, Japan).

Tube formulation assay
96-well culture plates (Costar, Corning) were coated with growth factor-reduced Matrigel (BD Biosciences) in a total volume of 30μl and allowed to solidify for 30 min at 37°C. BMVECs were trypsnized and resuspended at 5 × 10 4 cells/ml and 200 μl of this cell suspension were added into each well.
Tube formation was observed under an inverted microscope (Nikon, Japan).
Tube length was measured using the Image-Pro Plus 6.0 (Media Cybernetics, USA).

Bromodeoxyuridine incorporation
BMVECs were plated in 96-well plates at the density of 1 × 10 4 cells/well, and then subjected to growth arrest for 24 h before being exposed to OGD or treated with different agents in 5% FBS-DMEM. We measured Brdu incorporation according to the Millipore Brdu proliferation assay kit instruction.
Briefly, the cells were labeled with 10 ng/ml of Brdu during incubation, washed The cells were washed 3 times, and 100 μl substrates were added to each well and incubated for 30 min in darkness. Thereafter, the absorbance of samples was recorded at dual-wave lengths of 450 to 540 nm.

Western blotting
Proteins were extracted from BMVECs, using the procedures essentially the