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BackgroundNeuroinflammation and blood-brain barrier (BBB) disruption are two vital mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Recently, melanocortin-1 receptor (Mc1r) activation by Nle4-D-Phe7-α-MSH (NDP-MSH) was shown to play a neuroprotective role in an experimental autoimmune encephalomyelitis (EAE) mouse model. This study aimed to investigate whether NDP-MSH could alleviate neuroinflammation and BBB disruption after experimental ICH, as well as the potential mechanisms of its neuroprotective roles. MethodsTwo hundred and eighteen male C57BL/6 mice were subjected to autologous blood-injection ICH model.

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NDP-MSH, an agonist of Mc1r, was administered intraperitoneally injected at 1 h after ICH insult. To further explore the related protective mechanisms, Mc1r small interfering RNA (Mc1r siRNA) and nuclear receptor subfamily 4 group A member 1 (Nr4a1) siRNA were administered via intracerebroventricular (i.c.v) injection before ICH induction. Neurological test, BBB permeability, brain water content, immunofluorescence staining, and Western blot analysis were implemented. ResultsThe Expression of Mc1r was significantly increased after ICH. Mc1r was mainly expressed in microglia, astrocytes, and endothelial cells following ICH. Treatment with NDP-MSH remarkably improved neurological function and reduced BBB disruption, brain water content, and the number of microglia in the peri-hematoma tissue after ICH. Meanwhile, the administration of NDP-MSH significantly reduced the expression of p-NF-κB p65, IL-1β, TNF-α, and MMP-9 and increased the expression of p-CREB, Nr4a1, ZO-1, occludin, and Lama5.

Inversely, the knockdown of Mc1r or Nr4a1 abolished the neuroprotective effects of NDP-MSH. ConclusionsTaken together, NDP-MSH binding Mc1r attenuated neuroinflammation and BBB disruption and improved neurological deficits, at least in part through CREB/Nr4a1/NF-κB pathway after ICH.

Intracerebral hemorrhage (ICH) is a severe cerebral vascular disease with high morbidity and mortality, and its incidence is increasing annually. Mounting evidence has demonstrated that neuroinflammation and blood-brain barrier (BBB) disruption are two critical mechanisms of ICH-induced brain injury, which are closely associated with poor prognosis. Therefore, a therapeutic strategy targeting neuroinflammation and BBB disruption would be beneficial for attenuating brain injury following ICH.The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a member of the melanocortin family, a group of peptides derived from pro-opiomelanocortin (POMC). Α-MSH exerts well-established roles in the regulation of skin pigmentation and energy homeostasis, as well as inflammatory reaction ,. The biological function of α-MSH is mediated by five melanocortin receptors (termed Mc1r to Mc5r). Melanocortin-1 receptor (Mc1r), a G protein-coupled receptor, is best known as a mediator of the synthesis of melanin pigments, and it is also implicated in inflammation which is regulated by NF-κB signaling pathway ,. Α-MSH is released from cells in the central nervous system; however, the chemical property of α-MSH is unstable, transformed into the protease-stable Nle4-D-Phe7-α-MSH (NDP-MSH), which has a specific higher affinity to Mc1r ,.

Treatment with NDP-MSH was proven to reduce inflammation and vasospasm after subarachnoid hemorrhage. Likewise, the administration of NDP-MSH ameliorated blood-brain barrier (BBB) disruption by activating Mc1r in a model of experimental autoimmune encephalomyelitis (EAE). Despite the well-recognized roles of NDP-MSH and Mc1r on inflammation, the effects of NDP-MSH and Mc1r on neuroinflammation and BBB integrity after ICH have not been elucidated.Nuclear receptor subfamily 4 group A member 1 (Nr4a1), a member of Nur nuclear receptor family of transcriptional factors, is involved in neuroinflammation as a regulator of microglia activation in EAE in mice.

A previous study indicated that Nr4a1 was induced and functions immediately downstream of Mc1r signaling in melanocytic cells. Furthermore, Mykicki et al. Showed that NDP-MSH binding to Mc1r initiated the phosphorylation of cAMP response element-binding protein (CREB), and activated Nr4a1, subsequently exerted long-lasting neuroprotective roles in mice with EAE. It was reported that Nr4a orphan receptors could regulate NF-κB signaling in microglial and myeloid cells ,. Moreover, mounting evidence revealed that Nr4a1 negatively modulated the transcriptional activity of NF-κB and inhibited inflammatory gene expression ,.In the present study, we hypothesized that Mc1r activation by NDP-MSH could attenuate neuroinflammation and preserve BBB integrity after experimental ICH, and the protective mechanism is mediated through CREB/Nr4a1/NF-κB pathway. AnimalsAll experimental protocols for this study were approved by the Animal Ethics Committee of Chongqing Medical University. The study complied with the National Institutes of Health guide for the care and use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

A total of 218 C57BL/6 mice (male, weight about 25 g) were purchased from and bred at the Animal Center of Chongqing Medical University. All mice were housed in a light- and temperature-controlled room with free access to food and water. Experimental designFour separate experiments were designed as follows (Fig. ). A total of 218 mice were used (Additional file: Table S1). Experiment 1The time course of endogenous Mc1r in the peri-hematoma tissue was measured by Western blot. The cellular localization of Mc1r was detected by double immunofluorescence staining at 24 h after ICH.

Experiment 2To evaluate the effects of NDP-MSH on neuroinflammation and BBB integrity, three doses of NDP-MSH (1.5, 5, 15 μg/mouse, Anaspec, USA) dissolved in phosphate-buffered saline (PBS) were administered intraperitoneally at 1 h after ICH insult. Mice were randomly divided into five groups: sham, ICH + vehicle (PBS), ICH + NDP-MSH (1.5 μg/mouse), ICH + NDP-MSH (5 μg/mouse), and ICH + NDP-MSH (15 μg/mouse). Neurological test and brain water content were examined at 24 and 72 h after ICH. Evans blue (EB) extravasation was evaluated at 24 h after ICH. Experiment 3To assess the effect of in vivo knockdown of Mc1r on neuroinflammation and BBB permeability, Mc1r small interfering RNA (siRNA) was administered by intracerebroventricular (i.c.v) injection at 48 h before ICH induction, and then followed with NDP-MSH (5 μg/mouse) treatment at 1 h after ICH. Neurological test, brain water content, EB extravasation, immunofluorescence staining, and Western blot were carried out at 24 h post-ICH.

Mice were randomly divided into five groups: sham, ICH + vehicle (PBS), ICH + NDP-MSH (5 μg/mouse), ICH + NDP-MSH (5 μg/mouse) + scrambled siRNA (Scr siRNA), and ICH + NDP-MSH (5 μg/mouse) + Mc1r siRNA. In addition, to verify the knockdown efficiency of Mc1r siRNA, the expression of Mc1r in the right hemisphere was analyzed by Western blot.

Mice were randomly assigned to four groups: Naive+Scr siRNA, Naive+Mc1r siRNA, ICH + Scr siRNA, and ICH + Mc1r siRNA. Experiment 4To investigate the underlying mechanisms of NDP-MSH-mediated neuroprotective effects, Nr4a1 siRNA was administered by i.c.v injection at 48 h before ICH induction, and then followed with NDP-MSH (5 μg/mouse) treatment at 1 h after ICH. Neurological test, brain water content, and Western blot were implemented at 24 h following ICH. Mice were randomly allotted into five groups: sham, ICH + vehicle, ICH + NDP-MSH (5 μg/mouse), ICH + NDP-MSH (5 μg/mouse) + Scr siRNA, and ICH + DNP-MSH (5 μg/mouse) + Nr4a1 siRNA. Moreover, to validate the knockdown efficiency of Nr4a1 siRNA, the expression of Nr4a1 was measured by Western blot.

Mice were randomly assigned to four groups: Naive + Scr siRNA, Naive + Nr4a1 siRNA, ICH + Scr siRNA, and ICH + Nr4a1 siRNA. ICH mouse model inductionThe ICH model was induced by autologous blood injection as previously described. Briefly, the mice were anesthetized and fixed prone in a stereotaxic frame. Drill a small hole about 1 mm in diameter at 2 mm to the right of the bregma. Then 30 μl autologous arterial blood without anticoagulation was drawn from the central artery of the tail and delivered into the basal ganglion (stereotaxic coordinates: 0.2 mm anterior, 2.3 mm right lateral to the bregma, and 3.5 mm ventral to the skull). Firstly, 5 μl of blood was injected at 0.7 mm above the target position.

Five minutes later, the remaining 25 μl blood was delivered at 3.5 mm depth. The needle was left for 10 min more after injection and withdrawn slowly at a rate of 1 mm/min. Bone wax was then applied to cover the drilled hole. The sham-operated animals were delivered an equal volume of sterile saline at the same position. Intracerebroventricular injectionIntracerebroventricular injection was performed as previously described. Briefly, mice were anesthetized and placed in a stereotactic head frame in the prone position.

A longitudinal incision was made along the midline and a burr hole was drilled to the right of the bregma (1.0 mm lateral of the bregma). Following the manufacturer’s instructions, Mc1r siRNA (Thermo Fisher Scientific, USA, MSS275666, GCG AUU CUG UAU GCC CAC AUG UUC A, UGA ACA UGU GGG CAU ACA GAA UCG C), Nr4a1 siRNA (Thermo Fisher Scientific, USA, MSS205160, GAA GAU GCC GGU GAC GUG CAA CAA U, AUU GUU GCA CGU CAC CGG CAU CUU C), or scramble siRNA was dissolved in sterile RNase-free water. Mc1r siRNA mixture or scramble siRNA (100 pmol/2 μl) was delivered into the ipsilateral ventricle at the depth of 2.5 mm. The needle was left for an additional 5 min after injection to avert possible leakage and was slowly withdrawn at a rate of 1 mm/min. The burr hole was sealed with bone wax, and the incision was closed with sutures.

Mice were placed in an individual recovery cage. Neurobehavioral function testNeurobehavioral functions were evaluated using the modified Garcia test and corner turn test at 24 or 72 h following ICH by a blinded investigator as previously described. In the modified Garcia test, seven items including spontaneous activity, axial sensation, vibrissae touch, limb symmetry, lateral turning, forelimb walking, and climbing were tested. In the corner turn test, mice were allowed to approach a 30° corner. The mice exited the corner with either a right turn or left turn.

Ten trials were performed, with at least a 30-s break between the trials. The percentage of a right turn to 10 trials was then calculated. BBB permeabilityTo evaluate BBB permeability, Evans blue (Aladdin, China) was injected intraperitoneally (100 μl of 4% solution in saline) as previously described with a slight modification.

After 3 h circulation, mice were transcardially perfused with cold phosphate-buffered saline (0.1 M, PBS, pH 7.4) under deep anesthesia. Afterwards, the brain was removed and divided into left and right hemispheres and stored at − 80 °C immediately. The right part of the brain was homogenized in 1100 μl PBS, sonicated, and centrifuged (12,000 g, 4 °C, 30 min). The supernatant was collected and added an equal amount of trichloroacetic acid (TCA) to incubate overnight by 4 °C. After centrifugation (12,000 g, 4 °C, 30 min), Evans blue stain was measured by spectrophotometer (Thermo Fisher Scientific, USA) at 610 nm.

Brain water contentBrain water content was measured at 24 h and 72 h after ICH by an investigator blind to group information as previously described. In short, mice were sacrificed under deep anesthesia. The brain was immediately removed and cut into 4 mm coronal slice. The brain slice was separated into five parts: ipsilateral and contralateral basal ganglia, ipsilateral and contralateral cortex, and cerebellum. The cerebellum was retained as an internal control. Each part was immediately weighed on an electronic analytical balance (FA2204B, Techcomp, USA) to determine the wet weight (WW) and then dried at 100 °C for 72 h to determine the dry weight (DW).

Brain water content (percentage) was calculated as (WW − DW)/WW × 100%. Immunofluorescence stainingDouble fluorescence staining was performed as described previously. The mice were deeply anesthetized and were transcardially perfused with 20 ml ice-cold PBS followed by 20 ml of 4% paraformaldehyde at 24 h post-ICH. The whole brain was collected and then fixed in 4% paraformaldehyde for another 24 h. Afterwards, the brain was fixed in 20% sucrose solution until the tissue sink to the bottom followed by 30% sucrose solution for another 24 h. After being frozen at − 25 °C, the brain was cut into 10-μm-thick coronal sections using a cryostat (CM1860; Leica Microsystems, Germany).

To conduct double immunohistochemistry staining, the brain sections were incubated with primary antibody of anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100, Abcam, ab153696), anti-glial fibrillary acidic protein (GFAP, 1:200, CST, 3670, AB561049), anti-von Willebrand factor (vWF, 1:50, Santa Cruz, sc-365712, AB10842026), anti-NeuN (1:100, Abcam, ab104224, AB10711040), and anti-Mc1r (1:50, Genetex, GTX108190) overnight at 4 °C. After being incubated with the appropriate secondary antibody (1:200, Bioss) at 37 °C for 1 h, the sections were visualized and photographed with a fluorescence microscope (U-HGLGPS, OLYMPUS, Japan). Microphotographs were analyzed with cellSens Standard software. The numbers of Iba-1-positive cells were identified and counted in three different fields in peri-hematoma area from five random coronal sections per brain, and data were expressed as cells/field. Western blottingAfter mice were perfused with ice-cold PBS (0.1 M, pH 7.4) at 24 h post-operation, the peri-hematoma tissues were collected and stored in − 80 °C freezer until use. Western blotting was performed as previously described. After sample preparation, equal amounts of protein were loaded onto an SDS-PAGE gel.

After being electrophoresed and transferred to a PVDF membrane, the membrane was blocked 2 h at 37 °C followed by incubated with the primary antibody overnight at 4 °C. Mortality and exclusionThe total mortality of ICH mice was 9.34% (17/182) in this study. None of the sham group mice died. There was no significant difference in mortality rate among the experimental groups. Six mice were ruled out from this study due to no hemorrhage (Additional file: Table S1).

Expression of Mc1r after ICHAs shown in Fig. a, the Mc1r expression in the peri-hematoma tissue was significantly increased at 24 h and reached its peak at 72 h after ICH, when compared to the sham group. Double immunofluorescence staining showed that Mc1r was mainly expressed in the microglia, astrocytes, and endothelial cells in the peri-hematoma tissue at 24 h after ICH (Fig. c). Expression of Mc1r after intracerebral hemorrhage (ICH). A Representative Western blot band and quantitative analyses of Mc1r time-dependent expression from the peri-hematoma tissue after ICH. Administration of NDP-MSH improved neurological deficits and reduced brain edema and BBB permeability after ICHThe neurological deficits and brain edema were evidently worse at 24 and 72 h post-ICH in the ICH + vehicle and ICH + NDP-MSH (1.5 μg/mouse) groups, when compared with sham group. However, the administration of NDP-MSH (5 μg/mouse) and NDP-MSH (15 μg/mouse) significantly improved the neurological deficits (Fig. a, b) and reduced brain edema in ipsilateral basal ganglion (Fig. c).

Based on these results, the optimal dose of NDP-MSH was 5 μg/mouse, which was used for the rest of the experiments. BBB permeability was assessed by EB extravasation in the right cerebral hemispheres. EB extravasation in the ICH + vehicle group was significantly increased at 24 h after ICH, whereas NDP-MSH treatment (5 μg/mouse) prominently decreased EB dye leakage compared with the ICH + vehicle group (Fig. d). The neuroprotective effects of NDP-MSH on neurological functions, brain water content, and blood-brain barrier permeability after intracerebral hemorrhage (ICH). Treatment with NDP-MSH significantly improved neurological deficits ( a and b) and reduced brain water content ( c) at 24 and 72 h, as well as decreased EB extravasation at 24 h after ICH ( d).

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N = 6 for each group. Brain sections were divided into five parts: ipsilateral basal ganglia (ipsi-BG), contralateral basal ganglia (contra-BG), ipsilateral cortex (ipsi-CX), contralateral cortex (contra-CX), and cerebellum. Mc1r in vivo knockdown aggravated neurological deficits, brain edema, and BBB disruption after ICHTo further investigate, the protective role of NDP-MSH and Mc1r siRNA was administered by i.c.v injection to knockdown the expression of endogenous Mc1r.

Western blot showed that the Mc1r expression was inhibited by Mc1r siRNA at 72 h after injection (Fig. a). The knockdown of Mc1r abolished the protective effect of NDP-MSH on neurological functions (Fig. b, c), brain edema (Fig. d), and BBB integrity (Fig. e) at 24 h post-ICH. The effect of Mc1r siRNA on neurological functions, brain water content and BBB integrity at 24 h after ICH. A The expression of Mc1r was obviously reduced in the right hemisphere by Mc1r siRNA at 24 h post-ICH.