Neurokinin Receptor 1 (NK1R) Antagonist Aprepitant Enhances Hematoma Clearance by Regulating Microglial Polarization via PKC/p38MAPK/NFκB Pathway After Experimental Intracerebral Hemorrhage in Mice

Abstract

Hematoma clearance is an important therapeutic target to improve outcome following intracerebral hemorrhage (ICH). Recent studies showed that Neurokinin receptor-1 (NK1R) inhibition exerts protective effects in various neurological disease models, but its role in ICH has not been explored. The objective of this study was to investigate the role of NK1R and its relation to hematoma clearance after ICH using an autologous blood injection mouse model. A total of 332 adult male CD1 mice were used. We found that the expression levels of NK1R and its endogenous ligand, substance P (SP), were significantly upregulated after ICH. Intraperitoneal administration of the NK1R selective antagonist, Aprepitant, significantly improved neurobehavior, reduced hematoma volume and hemoglobin levels after ICH, and promoted microglia polarization towards M2 phenotype. Aprepitant decreased phosphorylated PKC, p38MAPK, and NFκB p65, and downregulated M1 markers while upregulating M2 markers after ICH. Intracerebroventricular administration of the NK1R agonist, GR73632 or PKC agonist, phorbol 12-myristate 13-acetate (PMA) reversed the effects of Aprepitant. To demonstrate the upstream mediator of NK1R activation, we performed thrombin injection and found that it increased SP. Inhibiting thrombin suppressed SP and decreased M1 markers while increasing M2 microglia polarization. Thus, NK1R inhibition promoted hematoma clearance after ICH by increasing M2 microglial polarization via downregulating PKC/p38MAPK/NFκB signaling pathway, and thrombin may be a key upstream mediator of NK1R activation. Therapeutic interventions inhibiting NK1R signaling may be a new target for the treatment of ICH.

Keywords: Hematoma clearance; Intracerebral hemorrhage; Microglia polarization; Neurokinin receptor-1; Thrombin.

Fig. 1

Time course expression of substance P and NK1R after ICH, and cellular location of NK1R after ICH. A–C Representative western blot bands and quantitative analyses of substance P (SP) and NK1R expression in the ipsilateral hemisphere in sham and ICH mice. Data was expressed as mean ± SD. *p < 0.05 vs sham; one-way ANOVA, Tukey test, n = 6/group. D Representative pictures of immunofluorescence staining for NK1R (green) expression in microglia (Iba-1, red), neurons (NeuN, red), and astrocytes (GFAP, red) in sham group and the peri-hematoma area 24 h after ICH. Scale bar = 50 μm. n = 2

Fig. 2

The effects of Aprepitant administration on hematoma volume, hemoglobin levels, brain edema, and neurobehavior tests at 24-h post-ICH. A Representative photograph of brain sections, B–C quantitative analyses of hematoma volume and hemoglobin level, D brain water content, E modified Garcia test, F corner turn test, and G forelimb placement test at 24 h after ICH. Data was represented as mean ± SD. *p < 0.05 vs sham; @p < 0.05 vs ICH + vehicle; #p < 0.05 vs ICH + Aprepitant 40 mg/kg; &p < 0.05 vs Aprepitant 360 mg/kg. One-way ANOVA, Tukey test, n = 6/group. RB right basal ganglia, RC right cortex, LB left basal ganglia, LC left cortex, CB cerebellum

Fig. 3

Effects of Aprepitant administration at different time-points after ICH and activation of NK1R by GR73632 on ICH outcomes evaluated at 72-h post-ICH. A, H Representative photograph of brain sections, B–C, I–J quantitative analyses of hematoma volume and hemoglobin level, D brain water content, E modified Garcia test, F corner turn test, and G forelimb placement test at 72 h after ICH. Data was represented as mean ± SD. *p < 0.05 vs sham; @p < 0.05 vs ICH + vehicle. One-way ANOVA, Tukey test, n = 6/group. RB right basal ganglia, RC right cortex, LB left basal ganglia, LC left cortex, CB cerebellum

Fig. 4

The effects of Aprepitant administration on hematoma volume, hemoglobin levels, and neurobehavior tests at 7 days post-ICH. A Representative photograph of brain sections, B–C quantitative analyses of hematoma volume and hemoglobin level, D Modified Garcia test, E corner turn test, and F forelimb placement test at 7 days after ICH. *p < 0.05 vs sham; @p < 0.05 vs ICH + vehicle. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n = 6/group

Fig. 5

Effects of NK1R inhibition with Aprepitant on long-term outcomes after ICH. A–B Nissl staining representative micrographs and neuronal quantification of the hippocampal CA1, CA3, and DG regions; C water maze test representative heat maps during probe trial; D probe quadrant duration on day 28 after ICH; E swimming distance and F escape latency on days 23 to 27 after ICH; and G foot fault test and H rotarod test at second and third week post-ICH. Data was represented as mean ± SD. *p < 0.05 vs sham; @p < 0.05 vs ICH + vehicle group; One-way ANOVA, Tukey post hoc test (B and D). Two-way repeated measures ANOVA, Tukey post hoc test (E–H), n = 15/group

Fig. 6

Effects of NK1R inhibition with Aprepitant on microglia polarization at 72 h after ICH. A Representative images of immunofluorescence staining of CD16 (green), CD86 (green), CD206 (green), and CD163 (green) with Iba-1 (red) in the perihematomal area at 72 h after ICH; B schematic illustration of brain tissue shows the perihematomal region depicted with a white square; C–F quantitative analyses of CD16, CD86, CD206, and CD163-positive cells in the perihematomal area at 72 h after ICH. Data was represented as mean ± SD. *p < 0.05 vs sham, @p < 0.05 vs ICH + vehicle. One-way ANOVA, Tukey test, n = 6/group

Fig. 7

Activation of NK1R or PKC abolished the effects of Aprepitant after ICH. A Representative western blot bands; B–I quantitative analyses of NK1R, p-PKC/PKC, p-P38/P38MAPK, p-P65/P65 NFκB, CD206, CD163, CD16, and CD86 in the ipsilateral hemisphere at 72 h after ICH. Data was represented as mean ± SD. *p < 0.05 vs Sham, @p < 0.05 vs ICH + vehicle, #p < 0.05 vs ICH + Aprepitant + DMSO; one-way ANOVA, Tukey post hoc test, n = 6/group

Fig. 8

Thrombin regulated NK1R activation by promoting substance P release and effects of hirudin was reversed with NK1R agonist GR73632. A, C, E Representative western blot bands; B, D, F quantitative analyses of substance P (SP), NK1R, CD206, CD163, CD16, and CD86 in the ipsilateral hemisphere at 72 h after surgery; G modified Garcia test; H corner turn test; and I forelimb placement test at 72 h after ICH. Data was represented as mean ± SD. *p < 0.05 vs Sham, &p < 0.05 vs thrombin + PBS, %p < 0.05 vs thrombin + DMSO; @p < 0.05 vs ICH + PBS, $p < 0.05 vs ICH + Hirudin + DMSO, one-way ANOVA, Tukey post hoc test, n = 6/group