CD200Fc Improves Neurological Function by Protecting the Blood–brain Barrier after Intracerebral Hemorrhage

Abstract

CD200 is widely distributed in the central nervous system and plays an essential role in the immune response in neurological diseases. However, little is currently known about the effects of CD200 signaling on the blood–brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of CD200 during ICH in an autologous blood induced mouse ICH model was investigated. Following ICH, critical protein expression, BBB permeability, and neurological function were measured with or without CD200Fc administration. Our results showed that both the expression of CD200 and CD200R1 decreased after ICH and administration of CD200Fc attenuated BBB leakage and improved neurological functions. In conclusion, our work demonstrated that CD200Fc might be a potential treatment option for ICH by protecting the BBB and improving functional outcomes.

Keywords

CD200 CD200R1 intracerebral hemorrhage blood–brain barrier

Introduction

Intracerebral hemorrhage (ICH) is associated with high mortality and high morbidity^1
–3. Currently, no effective treatment option is available to improve functional outcomes in patients with ICH. Beyond the immediate impact of ICH, secondary brain injury is the leading cause of death and long-term disability following ICH⁴. Blood–brain barrier (BBB) disruption, which leads to tissue exudation, neuronal necrosis, and apoptosis, further propagates brain edema⁵. The combination of such pathologies has been noted as an essential aspect of secondary brain injury.

CD200 is a type-I cell surface glycoprotein. It interacts with its receptor CD200 R, which includes CD200R1, CD200R2, CD200R3, and CD200R4 in mice and is expressed on myeloid cells (including microglia). The CD200 R family is responsible for the regulation of microglia function in several neurological diseases, such as Parkinson’s disease⁶, multiple sclerosis⁷, white matter ischemia⁸, and spina bifida⁹. The main isoforms of the CD200 R family which are expressed in the mouse brain are CD200R1 and R4. While the human CD200 R family only contain CD200R1 and CD200R2¹⁰. Thus, we focused our attention on CD200R1 in this study. CD200Fc, a fusion protein of CD200 and the immunoglobulin (Ig)G1 Fc segment applied in this study was reported to interact specifically with CD200R1 but not CD200R2-R4^11
–13.

In previous studies which utilized a CD200 knockout mice model, higher BBB permeability and more infiltration of peripheral proinflammatory cells in the nervous system was observed¹⁴, which indicated that CD200 might be related to BBB function and neuroinflammation.

In this study, we aimed to explore whether CD200Fc treatment can attenuate ICH-induced BBB disruption.

Materials and Methods

Animals

Animal protocols were approved by the Zhejiang University Committee on the Use and Care of Animals. A total of 124 male C57/Bl6 mice (6.5% mortality or asymptomatic) weighing 20–24 g were used in this study.

ICH Models

The homologous blood injection ICH model was adapted from a previously described model in mice¹⁵. The animals were anesthetized with 1% pentobarbital (80 mg/kg, intraperitoneally), and then positioned in a stereotaxic frame (World Precision Instruments, Sarasota, FL, USA), while core temperature was maintained at 37.5°C with a heating pad (temperature was monitored with a rectal thermometer). A cranial hole (1 mm) was drilled 2.2 mm lateral and 0.2 mm anterior to the bregma, near the right coronal suture. Autologous blood was collected from the caudal artery. A 26-gauge needle was inserted to the right basal ganglia through the hole (3.5 mm ventral). A total of 20 μl of autologous blood was injected at a rate of 2 μl/min using an automated pump (World Precision Instruments). The needle remained fixed for 5 min to prevent reflux and then was gently removed to limit any unintended damage. Animals in the sham groups only had a needle inserted and then removed.

Experimental Design

The experiments were conducted as follows.

Experiment 1

The time course of CD200 and CD200R1 expression after ICH were analyzed using Western blot. The ipsilateral hemispheres of mice from group sham, 6 h, 12 h, 24 h, 72 h were collected for analysis.

Experiment 2

Immunofluorescence was performed to check the localization of CD200R1 at 24 h after ICH. Four mice were used in both the sham group and the ICH group.

Experiment 3

For the Evans blue test, 24 mice were randomly divided into sham, ICH, ICH + CD200Fc (0.08 μg/g, intracerebroventricularly (i.c.v)), and ICH + IgG (0.08 μg/g, i.c.v) groups (n=6). Treatments were administrated at 1 h after surgery. The function of the BBB in each group was assessed at 24 h using Evans blue. For the brain water content test, a total of 24 mice were divided and treated similarly. Brain edema was evaluated by measuring brain water content.

Experiment 4

Mice were randomly divided into four groups with six animals in each group: sham, ICH, ICH + CD200Fc (0.08 μg/g, i.c.v), and ICH + IgG (0.08 μg/g, i.c.v) groups. Neurobehavioral tests including the Garcia test, foot-fault, corner turn, and rota-rod were performed.

Experiment 5

The downstream molecule Dok1 was measured in sham, ICH, ICH + CD200Fc (0.08 μg/g, i.c.v), and ICH + IgG (0.08 μg/g, i.c.v) groups (n=4), using Western blot.

Western Blotting

Mice were transcardially perfused with 50 ml of ice-cold phosphate-buffered saline (PBS). The brain tissue was homogenized and then centrifuged at 12,000 r/min for 30 min at 4°C, and the supernatant was collected. Protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce, Thermo Scientific, Waltham, MA, USA). Protein was then loaded and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose (NC) membrane. After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with primary antibodies: rabbit anti-CD200 (1:300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-CD200R1 (1:300, Santa Cruz), mouse anti-Dok1 (1:300, Santa Cruz) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:3000; KangChen, Shanghai, China). The membranes were then processed with a secondary antibody against goat IgG (1:3000; Multi Sciences, Hangzhou, Zhejiang, China) or mouse IgG (1:3000; Multi Sciences, China) for 2 h at room temperature. Bands were visualized using the chemiluminescence (ECL) detection reagents and then analyzed with ImageJ software.

Immunofluorescence Staining

Mice were perfused with 4% paraformaldehyde in 0.1 mol/l PBS (pH 7.4). The brains were kept in 4% paraformaldehyde for 12 h and then dehydrated in 30% sucrose for 2 to 3 days at 4°C. A series of 10-μm coronal brain sections (LEICA, CM3050 S, Buffalo Grove, IL, USA) were blocked with 5% donkey serum albumin for 2 h at room temperature and then incubated with goat anti-CD200R1 (1:200, R&D Systems, Minneapolis, MN, USA), rabbit anti-Iba-1 (1:500, Wako, Chuo-ku, Osaka, Japan), rabbit anti-GFAP (1:200, Abcam, Cambridge, MA, USA), and rabbit anti-NeuN (1:200, Abcam) at 4°C for 2 nights. After being rinsed three times with PBS, the sections were incubated with appropriate secondary antibodies for 2 h at room temperature. The perihematomal areas of stained sections were examined under a confocal laser scanning microscope (Olympus, FV3000, Tokyo, Japan). Microphotographs were analyzed with FV-10 ASW software.

Evans Blue Assay

Mice received an intraperitoneal injection of 2% Evans blue solution (4 ml/kg of body weight), at 24 h after surgery. Moreover, the stain was allowed to circulate for 3 h. Following circulation, mice were perfused with 50 ml ice-cold PBS. The brains were then removed and divided into left and right hemispheres, frozen in liquid nitrogen, and stored at −80°C. Right hemisphere samples were homogenized in PBS and then centrifuged at 12,000 r/min for 30 min at 4°C, and the supernatant was collected. An equal amount of 50% trichloroacetic acid was added to each 500 μl of the supernatants and then incubated overnight at 4°C. Later it was centrifuged at 12,000 r/min for 30 min at 4°C, and then the quantity of Evans blue stain was detected by spectrophotometer (BioTek, Winooski, VT, USA) at 610 nm and quantified according to a standard curve. These results are presented as milligrams of Evans blue stain per gram of brain tissue.

Brain Water Content

Brain water content was measured using the wet/dry method. Mice were sacrificed under anesthesia without perfusion at 24 h after surgery. The brains were removed quickly and divided into ipsilateral and contralateral cortices (Ipsi-CX and Cont-CX), ipsilateral and contralateral basal ganglia (Ipsi-BG and Cont-BG), and cerebellum (Cerebel). The wet weight of each part was measured immediately, and the dry weight was obtained after the brain tissue had been dried for 24 h.

Short-Term Neurobehavioral Test

Garcia test at 24 h after ICH was carried out, including assessment of spontaneous activity, axial sensation, vibrissae proprioception, symmetry of limb movement, lateral turning, forelimb walking, climbing, and grabbing.

Long-Term Neurobehavioral Test

Foot-fault, corner turn, and rota-rod tests were performed every 24 h in the first 7 days after ICH. Foot-fault: animals were placed on a horizontal 26 × 13 grid floor (wire diameter 1.3 cm) for 5 min. The number of foot faults of each forelimb were recorded using a camera (Logitech Webcam, Lausanne, Switzerland). Corner turn: the mice were placed in a 30-degree corner. We then recorded whether the animals turned left or right. The corner turn test was repeated 10 times for each mouse, and the percentage of right turns was calculated. Rota-rod: animals were placed on an accelerating rod (YLS-4C, Xuzhou, Jiangsu, China), with a max speed of 40 r/min and the time to fall was recorded for each run. All animals were trained twice daily until their results were consistent, before surgery.

Statistical Analysis

All data was given in means ± standard deviation (SD). The data was analyzed using GraphPad Prism version 6.02 with a Student’s t test and one-way analysis of variance (ANOVA). Multiple comparisons between the groups were performed using a Tukey test. Differences were considered significant at p < 0.05.

Results

Expression of CD200 and CD200R1 after ICH

Results of the Western blot showed that the expression of CD200 decreased at 24 h (Fig. 1B). While the expression of CD200R1 decreased since 6 h (p < 0.05, versus sham) and rose again at 72 h (Fig. 1A).

Fig. 1.

CD200R1 and CD200 expression in brain after ICH. (A) Western blot showed significant decrease of CD200R1 at 6 h, 12 h, and 24 h after ICH (∗p < 0.05 versus sham), and increased at 72 h (#p < 0.05, versus 24 h). (B) CD200 decreased at 24 h after ICH (∗p < 0.05 versus sham). N=4 mice/group. Values are expressed as mean ± SD.

Expression of CD200R1 in Perihematomal Tissue

Double immunofluorescence staining was performed in the basal ganglia area of sham and ICH groups. The staining found that CD200R1 was expressed on microglia in both sham and ICH groups (Fig. 2A and B), while CD200R1 was not expressed on astrocytes or neurons (Fig. 2C and D).

Fig. 2.

Localization of CD200R1 in brain. (A–B) CD200R1 was detected on Iba-1 positive cells in sham and ICH groups. (C-D) CD200R1 did not co-localize with GFAP or NeuN.

CD200Fc Protected BBB Integrity at 24 h after ICH

To examine whether CD200R1 plays a vital role in BBB protection after ICH, we tested the permeability of the BBB in mice with Evans blue and brain edema with brain water content. The results showed that the accumulation of Evans blue increased in our mice at 24 h after ICH, compared with sham. Mice injected with normal IgG showed no significant difference from the nontreatment group. Also, ICH mice that received CD200Fc treatment had a lower release of Evans blue than ICH with IgG. (Fig. 3A). The brain water content was higher in ICH mice than sham mice. Besides, mice that received CD200Fc treatment showed significant improvement in both ipsilateral basal ganglia and cortex after ICH. (Fig. 3B).

Fig. 3.

Evans blue assay and brain water content. (A) Accumulation of Evans blue increased at 24 h after ICH (∗∗p < 0.01 versus sham). Evans blue decreased in mice treated with CD200Fc (#p < 0.05 versus ICH). N=6 mice/group. (B) Brain water content showed significant increase in ipsilateral basal ganglia and cortex at 24 h after ICH while was reversed by CD200Fc treatment (∗∗p < 0.01 versus sham, #p < 0.05 versus ICH). N=6 mice/group. Values are expressed as mean ± SD.

CD200Fc Improved Neurobehavioral Outcomes

A 1 week a neurobehavior test was performed in order to see whether CD200Fc influenced the functional recovery after ICH. Foot-fault and corner turn tests showed a significant difference between ICH groups and CD200R1 treatment groups on the first 7 days after ICH (Fig. 4A and B). The rota-rod test showed a significant improvement in CD200Fc treatment group from day 2 to day 7 (Fig. 4C). The Garcia test showed neuronal function improvement at 24 h after ICH in the CD200Fc-treated group (Fig. 4D).

Fig. 4.

Neuronal behavior test at 24 h after ICH. (A–C) Long-term tests showed significant improvement in CD200Fc-treated mice. (D) Short-term Garcia test showed significance improvement in CD200Fc-treated mice. (∗p < 0.05 versus sham, ∗∗p < 0.01 versus sham, #p < 0.05 versus ICH, ##p < 0.01 versus ICH. Values are expressed as mean ± SD).

CD200Fc Upregulated Dok1

Dok1, the downstream protein of CD200R1, decreased at 24 h after ICH, which was reversed by treatment with CD200Fc. (Fig. 5)

Fig. 5.

Dok1 expression after ICH and different treatments. Dok1 expression decreased at 24 h after ICH. While treatment with CD200Fc showed significant improvement (∗∗p < 0.01 versus sham, ##p < 0.01 versus ICH. Values are expressed as mean ± SD).

Discussion

Though intracerebral hemorrhage is the leader of mortality and morbidity in all subtypes of stroke, there are few effective treatments for improving functional outcome in patients with ICH¹⁶. In this study, we found that administration of CD200Fc attenuated BBB disruption at 24 h and improved functional neurological outcomes of mice in the first week after ICH. CD200, the ligand of CD200 R family, is a surface protein widely expressed in central nervous system¹⁷. The decrease of interaction between CD200 and its receptor may promote the proinflammatory effect of microglia¹⁸. The decrease of CD200 expression and exaggeration of microglial response was reported in several disease models such as multiple sclerosis¹⁹ and Parkinson’s disease²⁰. Though a microglia response has been noted, little is known about the modification of the CD200 receptors. Studies have shown a decrease of CD200 R on macrophages in patients with Parkinson’s disease²¹ as well as on microglia in Alzheimer’s disease patients²². However, no significant alteration of CD200 R expression was found in patients with multiple sclerosis²³. In the present study, we found that both CD200 and its receptor decreased after ICH in mice. A recent study showed that the disruption of the BBB might reduce CD200 expression by interleukin (IL)-6 and tumor necrosis factor (TNF)-α produced by the microglia²⁴. Though CD200 expression is better understood, there is still a substantial gap in the understanding of why there would be a decrease in CD200R1. According to some studies, anti-inflammatory cytokines, such as IL-4 and IL-13 stimulate CD200 R expression on microglia^22,25. This stimulation may be related to the activation of microglia and ultimately responsible for BBB disruption and neurobehavior deficit after ICH.

The BBB is a system composed of microvascular endothelium, astrocytes, basement membrane, pericytes, and neurons^26,27. In a thrombin-induced ICH injury model, blocking the downstream activation of thrombin alleviated BBB disruption and promoted brain microvascular endothelial cell and astrocyte regeneration²⁸. This experimental design allows for a new approach for studying neuroprotection after cerebral hemorrhage and the pathologies of secondary brain injury.

Microglia get activated very soon after ICH and play a vital role in secondary injury^29,30. Yan et al.³¹, found that microglia can not only work on neurons or other glia but also alter the microvascular structure. As more evidence continues to suggest that microglia are powerful players in innate immunity and maintain homeostasis in the nervous system, microglia should not be separated from the BBB³⁰. When over-activated, microglia accumulate around the blood vessels, and destruction of microvascular structures takes place, including atrophy of endothelial cells, thickening of basement membrane, vacuolization, and degeneration of pericytes. The accumulation of these complications results in the changes of BBB permeability and exudation around the blood vessels^32,33. Previous studies have demonstrated that, CD200 is expressed mainly on the neurons, but also vascular endothelial cells⁶ and astrocytes³⁴. In the present study, we found CD200R1 is expressed on microglia but not on neurons or astrocytes, which indicated that the CD200–CD200R1 pathway might be a bridge between neurovascular units and microglia. Thus, further studies need to be carried out to determine whether CD200Fc protects the BBB or improves neurological functions through regulating microglia.

Since CD200 fusion protein has been used in several in vivo experiments^8,35
–37, we treated mice with CD200Fc at a dosage accordingly^36,37. Following treatment, Evans blue, brain water content and a series of neurobehavioral tests were applied to estimate the treatment effect of CD200Fc. We found that CD200Fc treatment resulted in a significant reduction of BBB permeability, Evans blue accumulation and brain water content was decreased in CD200Fc-treated mice, compared with the nontreatment group and the IgG-treated group. This finding coincided with the improvement of the neurobehavioral scores of the CD200Fc-treated mice. Among the long-term neurobehavioral tests, foot-fault and corner turn tests showed a significant difference following the first day after administration of CD200Fc, while the rota-rod test showed a significant difference following the second day.

There are a few limitations to our study. We directly focused on BBB function but did not look into the anti-inflammation or anti-apoptotic effect of CD200R1, although anti-apoptosis of CD200R1 function is quite widely studied in tumor and autoimmune diseases. Furthermore, the detailed mechanism about how the signaling pathways of CD200/CD200R1 changed with or without CD200Fc treatment in ICH was not demonstrated in our study. Previous studies have confirmed in both in vivo and in vitro studies using rat models, that when CD200 R binds to CD200, Y297 tyrosine in the PTB domain of its intracellular NPXY sequence is rapidly phosphorylated, then the phosphorylated inhibitory molecule Dok1 and Dok2 proteins are recruited and bound to Ras-GAP and SHIP. This cascade furtherly inhibits RAS and its downstream ERK, JNK, and P38 MAPK activation^38
–40. As a downstream molecule of ERK, MMP-9 has been proven to be important in BBB permeability^41
–43. Here we assume that activation of CD200 R protects BBB through ERK/MMP-9 signaling pathway. Thus, future studies are needed to investigate which downstream signals take charge in ICH and what changes take place during the treatment.

Footnotes

Ethical Approval

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Zhejiang University.

Statement of Human and Animal Rights

All procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee, Zhejiang University.

Statement of Informed Consent

There are no human subjects in this article, and informed consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (NSFC; 81471168) to Feng Gao, NSFC (81500991) to Lu-sha Tong.

ORCID iD

Chen-sheng Le

References

Tsai

Thomas

Sudlow

. Epidemiology of stroke and its subtypes in Chinese vs white populations: a systematic review. Neurology. 2013;81(3):264–272.

Poisson

Glidden

Johnston

Fullerton

. Deaths from stroke in US young adults, 1989-2009. Neurology. 2014;83(23):2110–2115.

Wang

Hoff

Yang

Liu

Wang

Zhao

. China National Stroke Registry (CNSR). Clinical characteristics, management, and functional outcomes in Chinese patients within the first year after intracerebral hemorrhage: analysis from China National Stroke Registry. CNS Neurosci Ther. 2012;18(9):773–780.

Lattanzi

Brigo

Trinka

Cagnetti

Di Napoli

Silvestrini

. Neutrophil-to-lymphocyte ratio in acute cerebral hemorrhage: a system review. Transl Stroke Res. 2019;10(2):137–145.

Aronowski

Zhao

. Molecular pathophysiology of cerebral hemorrhage: secondary brain injury. Stroke. 2011;42(6):1781–1786.

Wang

Zhang

Chen

. CD200-CD200 R regulation of microglia activation in the pathogenesis of Parkinson’s disease. J Neuroimmune Pharmacol. 2007;2(3):259–264.

Koning

Swaab

Hoek

Huitinga

. Distribution of the immune inhibitory molecules CD200 and CD200 R in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions. J Neuropathol Exp Neurol. 2009;68(2):159–167.

Hayakawa

Pham

Seo

Miyamoto

Maki

Terasaki

Sakadzic

Boas

van Leyen

Waeber

Kim

, et al. CD200 restrains macrophage attack on oligodendrocyte precursors via toll-like receptor 4 downregulation. J Cereb Blood Flow Metab. 2016;36(4):781–793.

Oria

Figueira

Scorletti

Sbragia

Owens

Pathak

Corona

Marotta

Encinas

Peiro

, et al. CD200-CD200 R imbalance correlates with microglia and pro-inflammatory activation in rat spinal cords exposed to amniotic fluid in retinoic acid-induced spina bifida. Sci Rep. 2018;8(1):10638.

10.

Gorczynski

Chen

Clark

Kai

Lee

Nachman

Wong

Marsden

. Structural and functional heterogeneity in the CD200 R family of immunoregulatory molecules and their expression at the feto-maternal interface. Am J Reprod Immunol. 2004;52(2):147–163.

11.

Liu

Bando

Vargas-Lowy

Elyaman

Khoury

Huang

Reif

Chitnis

. CD200R1 agonist attenuates mechanisms of chronic disease in a murine model of multiple sclerosis. J Neurosci. 2010;30(6):2025–2038.

12.

Wright

Cherwinski

Foster-Cuevas

Brooke

Puklavec

Bigler

Song

Jenmalm

Gorman

McClanahan

Liu

, et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol. 2003;171(6):3034–3046.

13.

Hatherley

Cherwinski

Moshref

Barclay

. Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor. J Immunol. 2005;175(4):2469–2474.

14.

Denieffe

Kelly

McDonald

Lyons

Lynch

. Classical activation of microglia in CD200-deficient mice is a consequence of blood brain barrier permeability and infiltration of peripheral cells. Brain Behav Immun. 2013;34:86–97.

15.

Wan

Cheng

Jin

Guo

Hua

Keep

. Microglia activation and polarization after intracerebral hemorrhage in mice: the role of protease-activated receptor-1. Transl Stroke Res. 2016;7(6):478–487.

16.

Jakubovic

Aviv

. Intracerebral hemorrhage: toward physiological imaging of hemorrhage risk in acute and chronic bleeding. Front Neurol. 2012;3:86.

17.

Hoek

Ruuls

Murphy

Wright

Goddard

Zurawski

Blom

Homola

Streit

Brown

Barclay

, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science. 2000;290(5497):1768–1771.

18.

Iadecola

Anrather

. The immunology of stroke: from mechanisms to translation. Nat Med. 2011;17(7):796–808.

19.

Valente

Serratosa

Perpina

Saura

Sola

. Alterations in CD200-CD200R1 system during EAE already manifest at presymptomatic stages. Front Cell Neurosci. 2017;11:129.

20.

Wang

Zhang

Yan

Zhao

Zhou

Wang

Zhang

. Impaired CD200-CD200R-mediated microglia silencing enhances midbrain dopaminergic neurodegeneration: roles of aging, superoxide, NADPH oxidase, and p38 MAPK. Free Radic Biol Med. 2011;50(9):1094–1106.

21.

Luo

Zhang

Liu

Zheng

Wang

Chen

Ding

. Altered regulation of CD200 receptor in monocyte-derived macrophages from individuals with Parkinson’s disease. Neurochem Res. 2010;35(4):540–547.

22.

Walker

Dalsing-Hernandez

Campbell

Lue

. Decreased expression of CD200 and CD200 receptor in Alzheimer’s disease: a potential mechanism leading to chronic inflammation. Exp Neurol. 2009;215(1):5–19.

23.

Koning

Hoek

Huitinga

. Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions. Ann Neurol. 2007;62(5):504–514.

24.

Singh

Kushwaha

Gera

Ansari

Mishra

Dewangan

Patnaik

Ghosh

. Sneaky entry of IFNgamma through arsenic-induced leaky blood-brain barrier reduces CD200 expression by microglial pro-inflammatory cytokine. Mol Neurobiol. 2019;56(2):1488–1499.

25.

Lyons

Downer

Crotty

Nolan

Mills

Lynch

. CD200 ligand receptor interaction modulates microglial activation in vivo and in vitro: a role for IL-4. J Neurosci. 2007;27(31):8309–8313.

26.

Persidsky

Ramirez

Haorah

Kanmogne

. Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol. 2006;1(3):223–236.

27.

Cai

Liu

Zhao

Chen

. Pericytes in brain injury and repair after ischemic stroke. Transl Stroke Res. 2017;8(2):107–121.

28.

Liu

Ander

Shen

Kaur

Deng

Sharp

. Blood-brain barrier breakdown and repair by Src after thrombin-induced injury. Ann Neurol. 2010;67(4):526–533.

29.

Wang

Dore

. Heme oxygenase-1 exacerbates early brain injury after intracerebral haemorrhage. Brain. 2007;130(Pt 6):1643–1652.

30.

Stanimirovic

Friedman

. Pathophysiology of the neurovascular unit: disease cause or consequence? J Cereb Blood Flow Metab. 2012;32(7):1207–1221.

31.

Yan

Lee

Lazzaro

Aranda

Grant

Chaqour

. Single and compound knock-outs of MicroRNA (miRNA)-155 and its angiogenic gene target CCN1 in mice alter vascular and neovascular growth in the retina via resident microglia. J Biol Chem. 2015;290(38):23264–23281.

32.

Nadeau

Dietrich

Wilkinson

Crawford

George

Nichol

Colbourne

. Prolonged blood-brain barrier injury occurs after experimental intracerebral hemorrhage and is not acutely associated with additional bleeding. Transl Stroke Res. 2019;10(3):287–297.

33.

Blecharz-Lang

Wagner

Fries

Nieminen-Kelha

Rosner

Schneider

Vajkoczy

. Interleukin 6-mediated endothelial barrier disturbances can be attenuated by blockade of the IL6 receptor expressed in brain microvascular endothelial cells. Transl Stroke Res. 2018;9(6):631–642.

34.

Dentesano

Serratosa

Tusell

Ramon

Valente

Saura

Sola

. CD200R1 and CD200 expression are regulated by PPAR-gamma in activated glial cells. Glia. 2014;62(6):982–998.

35.

Snelgrove

Goulding

Didierlaurent

Lyonga

Vekaria

Edwards

Gwyer

Sedgwick

Barclay

Hussell

. A critical function for CD200 in lung immune homeostasis and the severity of influenza infection. Nat Immunol. 2008;9(9):1074–1083.

36.

Cox

Carney

Miller

Lynch

. CD200 fusion protein decreases microglial activation in the hippocampus of aged rats. Brain Behav Immun. 2012;26(5):789–796.

37.

Hernangomez

Klusakova

Joukal

Hradilova-Svizenska

Guaza

Dubovy

. CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity immediately after its intrathecal application in a rat neuropathic pain model. J Neuroinflammation. 2016;13:43.

38.

Feng

Klebe

Ding

Guo

Z-N

Flores

Yin

Tang

Zhang

. Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage. J Cereb Blood Flow Metab. 2019;39(1):97–107.

39.

Mihrshahi

Barclay

Brown

. Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells. J Immunol. 2009;183(8):4879–4886.

40.

Zhang

Cherwinski

Sedgwick

Phillips

. Molecular mechanisms of CD200 inhibition of mast cell activation. J Immunol. 2004;173(11):6786–6793.

41.

Wang

Tsirka

. Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral haemorrhage. Brain. 2005;128(Pt 7):1622–1633.

42.

Wang

Luo

. Nano-curcumin simultaneously protects the blood-brain barrier and reduces M1 microglial activation during cerebral ischemia-reperfusion injury. ACS Appl Mater Interfaces. 2019;11(4):3763–3770.

43.

Qing-Feng

Ying-Peng

Tian-Tong

. Matrix metalloproteinase-9 and p53 involved in chronic fluorosis induced blood-brain barrier damage and neurocyte changes. Arch Med Sci. 2019;15(2):457–466.