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Immediate remote ischemic postconditioning reduces cerebral damage in ischemic stroke mice by enhancing leptomeningeal collateral circulation

Abstract Remote ischemic postconditioning (RIPC) is a promising neuroprotective strategy for ischemic stroke. Here, we employed a focal ischemic stroke mouse model to test the hypothesis that poststroke collateral circulation as a potent mechanism of action underlying the therapeutic effects of immediate RIPC. During reperfusion of cerebral ischemia, the mice were randomly assigned to receive RIPC, granulocyte colony‐stimulating factor (G‐CSF) as a positive control, or no treatment. At 24 hr, we found RIPC and G‐CSF increased monocytes/macrophages in the dorsal brain surface and in the spleen, coupled with enhanced leptomeningeal collateral flow compared with nontreatment group. Blood monocytes depletion by 5‐fluorouracil (5‐FU) significantly limited the neuroprotection of RIPC or G‐CSF treatment. The protein expression of proangiogenic factors such as Ang‐2 was increased by ischemia, but treatment with either RIPC or G‐CSF showed no further upregulation. Thus, immediate RIPC confers neuroprotection, in part, by enhancing leptomeningeal collateral circulation in a mouse model of ischemic stroke.

Here, using G-CSF as a positive control, we examined the effect of RIPC on monocyte recruitment and subsequent leptomeningeal collateral circulation in mice with ischemic stroke.

| Animal experimental protocol
Male C57BL/6J strain mice 9-10 weeks of age; Charles River Laboratories Inc., Beijing, China) were used in this study. The experimental procedures were approved by the Animal Care and Use Committee of Capital Medical University, China, and conducted according to National Institutes of Health guidelines.
Focal cerebral ischemia was generated on a modified 3-vessel occlusion (3VO) method as reported by Z. Liu et al. (2017). Briefly, general anesthesia was induced and maintained with 1.0-2.5% enfluane in 70% N 2 O and 30% O 2 (Bickford veterinary anesthesia equipment model no. 61010; AM Bickford Inc., Wales Center, NY). Under anesthesia, bilateral common carotid arteries (CCAs) were clipped for 15 min using surgical miniclips, whereas the stem of right distal middle cerebral artery (dMCA) was permanently cauterized using a bipolar coagulator (B. Braun Aesculap, Melsungen, Germany) through a 1-mmdiameter burr hole in the skull. Surgery was performed under a microscope (Carl Zeiss AG, Jena, Germany). A circulating heating pad and a heating lamp were used to keep rectal temperature at 37℃.
Sham-operated mice were exposed to the same conditions, but the dMCA and the CCAs were not manipulated. Stroke animals were randomly divided into three groups: (a) Stroke without treatment: 3VO followed by 24 hr of reperfusion; (b) Stroke&RIPC: RIPC was conducted immediately after CCAs release as described previously (Ren et al., 2011), generally by occluding and releasing the femoral arteries in bilateral lower limbs for three cycles; each occlusion/release lasted for 10 min; (c) Stroke&G-CSF: recombinant human G-CSF, 100 µg/kg body weight in 100 µl sterile saline, sc., immediately after the operation. All studies were performed in a blinded and randomized manner.

| Neurological testing
The corner test was used to examine stroke severity and functional deficits (Zhang et al., 2002). Each mouse was tested for 20 trials and the laterality index was calculated according to the formula: laterality index = (turns to the right side − turns to the left side)/total number of turns. A laterality index of 0 is indicative of animals presenting total symmetry. This test detects integrated sensorimotor function as it involves both stimulation of the vibrissae (sensory/neglect) and rearing (motor response).

| Infarct volume measurements
Twenty-four hours after surgery, mice were anesthetized with 10% chloral hydrate. Coronal brain sections with a 1-mm thickness were removed, and stained with 2% solution of 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich Corp., St. Louis, MO). To correct hemispheric edema, infarct volume of the ischemic region was normalized to the nonischemic region and expressed as a percentage of contralateral cortex volume.

| Visualization of vessels by latex perfusion
The leptomeningeal anastomoses were visualized by latex perfusion technique as previously reported (Woitzik, Hecht, Schneider, Peña-Tapia, & Vajkoczy, 2006). The mice were anesthetized with 1% enfluane in 70% N 2 O and 30% O 2 , and papaverine hydrochloride solution was slowly injected through the femoral vein at a rate of 100 µl/min (50 mg/kg body weight), then the right atrium of the heart was incised for the venous blood drain out. The left ventricle of the heart was cannulated and injected with 2 ml saline and subsequently 0.5 ml latex compound (Product No. 39210; UHU GmbH & Co. KG, Bühl/Baden, Germany), which was mixed with 50 µl/ml carbon black (Beijing Ink Product No. 1022-1144Beijing, China) beforehand. Then, the brain was removed carefully from the skull, and photographs of the dorsal and ventral brain surface were taken by a scanner (Scanjet G3110; HP, Shanghai, China).
The vessel diameters of the leptomeningeal anastomoses were measured at the point where the distal MCA joined the distal anterior cerebral artery (ACA). The vessel diameter of the Willis' circle was measured at the proximal point where the right ACA diverged the olfactory artery.
Images were captured using a microscope (Nikon, Tokyo, Japan).
To verify the monocyte depletion, blood analysis was performed by flow cytometry at 4 and 7 days after the injection during preliminary test.

| Western blot analysis
At 24 hr mice were transcardially perfused with 0.9% NaCl, and total protein was extracted from the bilateral cortices. Protein concentration determination and western blot analysis were performed as our previous report (Wei et al., 2016). The amount of proteins were quantified by densitometry and normalized to β-actin, an internal standard. Primary

| Enzyme-linked immunoassay (ELISA)
The levels of mouse G-CSF and human G-CSF in mouse plasma and brain cortex homogenate were determined by ultrasensitive ELISA kits (R&D Systems, Inc), according to the manufacturer's instructions.
Before assay, mouse plasma samples were 10-fold diluted whereas brain cortex homogenate two-fold, and 50 µL or 100 µL of diluted sample was added per well for mouse G-CSF or human G-CSF detection, respectively. Absorbance was read using a microplate reader set to 450 nm, and wavelength correction at 540 nm.

| Statistical analysis
All data were expressed as mean ± SEM. Statistical analysis was performed with SPSS for Windows (version 22, SPSS Inc., Armonk).
The differences among groups were assessed using one-way analysis of variance, followed by all pairwise multiple comparison procedures using the Bonferroni test. Statistical significance was set at p ≤ 0.05.

| Angioprotein-expression assay in ischemic cortex by western blot analysis
Ang-1 and Ang-2 were selected as markers of angiogenesis. There were no significant differences of Ang-1 expression between groups ( Figure 5a). Meanwhile ischemic insult upregulated Ang-2 expression in the ischemic cortex, whereas both treatments of RIPC and G-CSF showed no additional increase (Figure 5b).

| G-CSF levels in mouse brain and plasma
Both mouse G-CSF and human G-CSF levels in brain and serum from 20 mice were determined by ELISA (n = 5 per group). Mouse G-CSF in each contralateral cortex sample was 0 pg (Figure 6a

| DISCUSSION
The present study demonstrated that RIPC, which was conducted immediately after ischemic stroke in the bilateral hind limbs, of spleen-derived MMs in brain injury, has attracted great attention (Kim, Yang, Beltran, & Cho, 2014;Miró-Mur et al., 2016;Schmidt et al., 2017;Song et al., 2017). This study was designed to probe the effects of RIPC on leptomeningeal anastomoses growth, and our results support the concept of monocyte recruitment as playing a critical role in the enhancement of collateral flow (Heil et al., 2002;Sugiyama et al., 2011).
In addition to collateral flow, increased angiogenesis has been shown to promote neurological and histological improvement after stroke, in particular, demonstrating the outgrowth of new vessels from pre-existing vasculature (Dela Peña et al., 2015;J. Liu et al., 2014). In this study the angiogenesis marker Ang-1 and Ang-2 were examined at 24 hr after ischemic stroke. The results indicated that neither RIPC nor human G-CSF (100 µg/kg BW) treatment promoted angiogenesis via these factors. In a separate ongoing study, we are analyzing the angiogenic and neurogenic response by repeated daily RIPC in chronic phase after ischemic stroke. Recognizing that G-CSF could exert direct neuroprotective action, we inquired whether remote limb ischemic postconditioning could produce endogenous G-CSF into the peripheral blood and then into the brain. Using ultrasensitive ELISA, we examined mouse G-CSF levels in animal plasma and cerebral tissue to answer this question. The results refuted the hypothesis that endogenous G-CSF is mobilized by RIPC.
Nevertheless, considering the duration of the second protection window of ischemic conditioning (Hausenloy et al., 2016), the ability of repeated daily RIPC to produce accumulative effects to trigger endogenous G-CSF is worth future investigation.
Currently, the concept of remote ischemic conditioning-induced neuroprotection includes remote ischemic preconditioning (RI-PreC), remote ischemic perconditioning (RIPerC), and remote ischemic postconditioning (RIPostC) based on application timing either before ischemia, during ischemia, or during reperfusion of ischemia, respectively (Hess et al., 2013;Lim & Hausenloy, 2012;Wang et al., 2015). Our 3VO animal model represents a "mixed temporary/permanent" ischemia model that produces clear neocortical infarct and relevant to the most common type of human stroke.  F I G U R E 5 Western blot for angiopoietins Ang-1 and Ang-2. (a) There were no significant differences of Ang-1 expression between all the groups (n = 5). (b) Ischemic insult upregulated Ang-2 expression in the ischemic cortex, whereas both RIPC and G-CSF treatment showed no further increase (1.8-, 1.7-, 1.8-fold vs. Sham, *p < 0.05, n = 5). Data were expressed as mean ± SEM. Data were normalized to β-actin. G-CSF: granulocyte colony-stimulating factor; RIPC: remote ischemic postconditioning F I G U R E 6 Enzyme-linked immunoassay (ELISA) for mouse G-CSF. (a) Mouse G-CSF level in contralateral cortex of each mouse was 0, as well as the ipsilateral cortex of Sham group. There was an apparent increase of mouse G-CSF in the ipsilateral cortex in Stroke mice, whereas remote ischemic postconditioning and G-CSF significantly decreased the factor compared with Stroke without treatment (***p < 0.001 vs. Sham, ## p < 0.01, ### p < 0.001 vs. Stroke, n = 5). (b) Mouse G-CSF level in plasma of Stroke group showed significant decrease compared with Sham group (*p < 0.05), whereas RIPC and G-CSF treatment both attenuated the downtrend of mouse G-CSF in plasma caused by cerebral ischemic insult (n = 5). Data were expressed as mean ± SEM. G-CSF: granulocyte colony-stimulating factor RIC represents a highly translatable therapy for multiorgan protection against ischemia-reperfusion injury (IRI). A growing number of clinical trials have indicated that RIC exerts therapeutic effects, specifically in the heart (Cao, Zhang, Wang, Xia, & Yang, 2018;Cho et al., 2017) and brain (England et al., 2017;Pico et al., 2016). However, to date, a clear understanding of the mechanism through which RIC exerts its functional benefits remains a key hurdle in its clinical translation. Our present study showed that immediate RIPC reduced cerebral damage by leptomeningeal collateral flow augmentation via spleen-derived MMs recruitment in ischemic stroke mice. Combined with its noninvasive and safe profile, the collateral flow enhancement property of RIPC may facilitate its clinical application for ischemic stroke patients.