Pink1 deficiency enhances neurological deficits and inflammatory responses after intracerebral hemorrhage in mice

Pink1 (PTEN-induced putative kinase 1) is a protein associated with maintaining mitochondrial function and integrity and has been reported to mediate neurodegeneration and neuroinflammation. While the role of Pink1 in intracerebral hemorrhage (ICH)-related neurological deficits and inflammatory responses is not deciphered. Congenic blood was transfused into the left corpus striatum to construct the ICH model in C57/BL6 wild-type (WT) and Pink1−/− mice. The relative expression of Pink1, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-2, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, Cd86, nitric oxide synthase 2 (Nos2), Cd206, arginase 1 (Arg-1), and IL-10 was detected with qRT-PCR, Western blotting, or ELISA. Mouse neurological deficit scores (mNSS) and water content were detected, and an open-field test was performed to assay anxiety-like behavior. Remarkably decreased Pink1 expression and increased MIP-2, IL-1β, MCP-1, and TNF-α expression were observed after 12 h, 24 h, 48 h, 72 h, and 7 d post-ICH induction in the ipsilateral injury hemispheres. Pink1 deficiency could further up-regulate mNSS scores, brain water content, MIP-2, MCP-1, IL-1β, and TNF-α in the ipsilateral injury hemispheres. On the other hand, Pink1 deficiency could decrease the number of center cross, the velocity, and the total distance traveled in open field test. Pink1 deficiency could further up-regulate the mRNA levels of pro-inflammatory (M1) molecules (Cd86, Nos2), and down-regulate the relative expression of anti-inflammatory (M2) molecules (Cd206, Arg-1, and IL-10). Pink1 deficiency deteriorates neurological deficits and inflammatory responses after ICH, which can be considered as a treatment target.


Introduction
As the most devastating type of stroke, intracerebral hemorrhage (ICH) occurs at any age [1,2].The absorption and expansion of the resulting hematoma can trigger a series of reactions, which lead to primary and secondary brain injury [3].Mechanically, hematoma components may activate microglia and promote pro-inflammatory cytokines release to attract peripheral immune cell infiltration [4].Depending on the location, ICH-affected individuals may suffer from the abrupt onset of headache, nausea, partial paralysis, personality changes, or cognitive abilities alteration [5].In severe cases, ICH can cause permanent brain damage or death [6].Unfortunately, no clinically proven therapies are available, and the primary treatment is only supportive [7].
Increasing evidence demonstrates that innate inflammatory reactions might participate in short-term toxicity, neurological deficits, and longterm recovery [8,9].Considerable research has been performed to decipher the subsequent inflammatory cascade and to screen the potential therapeutic strategies [10][11][12].Activated and polarized microglia, either pro-inflammatory or anti-inflammatory phenotypes, are identified to be critical in neural repair and inflammation inhibition processes [13].
Our previous investigation demonstrates that PTEN-induced putative kinase 1 (Pink1) promotes microglia mitochondrial autophagy to protect against ICH-induced brain injury [14].In order to further study the significance of Pink1 in ICH and its relationship with ICH-induced neurological dysfunction and neuro-inflammation, time course neuro-inflammation molecule expression is detected.Pink1 deficiency mice are utilized to confirm the neurological protection and inflammatory inhibition role of Pink1 in ICH.

ICH model
C57BL/6J wild-type and C57BL/6J-Pink1 em1Cya (KOCMP-68943-Pink1-B6J-VA, Pink1 À/À ) mice purchased from Cyagen Saiye Biology (eight-week-old; male) were cultured in pathogen-free condition.Mice were anesthetized with intraperitoneal pentobarbital at a dose of 50 mg/ kg and immobilized on a Model 1430 stereotaxic frame (Kopf Instruments).A stainless steel cannula (26-g) was used to transfuse congenic tail vein blood into the left corpus striatum with a burr hole (1 mm diameter) on the skull at the speed of 15 μL/5 min.In the sham group, a burr hole on the skull was drilled without congenic tail vein blood transfusion.The timeline of tissue collection (Pre-surgery, 12 h, 24 h, 48 h, 72 h, and 7 d post ICH; n ¼ 26) to detect the relative expression and neurological deficit scores detection (Pre-surgery, 1 d, 3 d, 7 d, and 14 d post ICH; n ¼ 8) were indicated in the supplementary Figure S1.Animal studies were approved by the ethics committee of the Second Hospital of Hebei Medical University.

Western blotting
Ipsilateral injury hemispheres were isolated and lysed with Cell Lysis Buffer (Beyotime Biotechnology, Shanghai, China), and the soluble supernatants (20 μg) were loaded on 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes.After blocking with 5 % dry milk, the membranes were incubated with the primary antibody against Pink1 (sc-517353, Santa Cruz, Dallas, TX) at a 1:500 dilution and cleaved caspase 3 (ab214430, Abcam) at a 1:1000 dilution at 4 C overnight and peroxidase-conjugated secondary antibody (Santa Cruz) at a 1:2000 dilution for 2 h, which was developed with a GE Healthcare ECL system (Tanon).GAPDH (1:1000, sc-365062, Santa Cruz) was utilized as an internal control.

Water content
The degree of brain edema was revealed by the water content with wet/dry method.After the animals were anesthetized, the brain tissues were taken out immediately, and thick brain slices (4 mm) were cut around the injection marks, which were weighed with a Sartorius electronic balance as wet weight (WW).The weighed slides were further roasted at 100 C for 24 h and weighed as dry weight (DW).The formula of [WW À DW]/WW Â 100 % was calculated as the water content of brain tissue (%).

Open field test
Locomotion activity was assayed in a light-grey polyvinyl chloride activity chamber (55 Â 55 Â 36 cm), which was divided into four quadrants.After placing mice in the center of the chamber, locomotion activity was recorded for 5 min (n ¼ 12 per group).The total time in the center zone, the frequency of the center cross, time spent near the walls, and outside the center were measured.Distance traveled, velocity, and reduced frequency of center cross were utilized as indications of anxiety behavior or locomotor activity.

Statistical analysis
Data was shown with mean AE standard deviation (SD).One-way ANOVA followed Dunn's multiple comparisons test or two-way ANOVA followed Tukey's multiple comparisons test was performed.Image J was utilized for quantitative analysis.The significance level was set as p < 0.05 for both ANOVA and Spearman's correlation analysis.

Pink1 negatively correlates with inflammatory cytokine expression
Time course analysis was utilized to reveal the expression of Pink1 after congenic blood infusion in the hemispheres.The relative mRNA (Fig. 1A) and protein (Fig. 1B and C) levels of Pink1 in the ipsilateral injury hemispheres of wild-type mice were detected, as expected, remarkably decreased Pink1 expression was observed after 12 h, 24 h, 48 h, 72 h, and 7 d post ICH.It was worth noting that the relative expression of Pink1 demonstrated time-dependent decrease trends at the acute phase (0-72 h), while on 7 d, a plateau occurred.

Pink1 deficiency enhances neurological deficits and brain edema
The phenotype of Pink1 wide-type mice and Pink1 deficiency mice was confirmed by Western blotting detection (supplementary Figure S2).On 1, 3, 7, and 14 days after ICH induction, mNSS scores peaked on day 1, and then decreased in both Pink1 wide-type mice and Pink1 deficiency mice (Fig. 4A).At the same time, increased brain water content was measured 3 days after ICH induction in both wide-type mice and Pink1 deficiency mice (Fig. 4B).All of these indicated the success of ICH model construction.On the other hand, Pink1 deficiency further up-regulated mNSS scores (Fig. 4A, p < 0.05), brain water content (Fig. 4B, p < 0.05), and the relative expression of cleaved Caspase 3 (supplementary Figure S3A and S3B).These results testified that Pink1 deficiency enhanced brain edema, neurological deficits, and apoptosis caused by ICH.

Pink1 deficiency promotes anxiety-like behavior
ICH induction could increase immobility time (Fig. 5A), as well as decrease the number of the center cross (Fig. 5B), the total distance traveled (Fig. 5C), and the velocity (Fig. 5D) in both wide-type and Pink1 deficient mice.On the other hand, Pink1 deficiency further increased the immobility time (Fig. 5A, p < 0.01) and decreased the number of center cross (Fig. 5B, p < 0.05), the total distance traveled (Fig. 5C, p < 0.05), and the velocity (Fig. 5D, p < 0.01).All of these data indicated that Pink1 deficiency could promote the anxiety of mice caused by ICH.

Discussion
Although significant progress has been made, ICH is still a serious public health problem in adults worldwide with neurological deficits and    In this investigation, we demonstrate that Pink1 deficiency could enhance inflammatory responses in the ipsilateral injury hemispheres.It must be mentioned that both central nervous system-intrinsic microglia and monocyte-derived macrophages might contribute to the development of ICH.Although the precise cellular source of pro-inflammationand anti-inflammation-associated gene expression in ICH is not indicated, intrinsic microglia or monocyte-derived macrophages is assumed as the main cellular source.
As resident immune cells in the central nervous system, microglia not only remove the hematoma and clear debris but also make M1 and M2 phenotype change to produce pro-inflammatory cytokines and neuroprotective anti-inflammatory cytokines to determine the progression of ICH [13,[17][18][19].In LPS/IFN-γ stimulated inflammation, Pink1 deficiency can attenuate expression of both pro-inflammatory and anti-inflammatory cytokines in microglia, while enhancing pro-inflammatory phenotypes of mixed astrocytes/microglia and pure astrocytes [20].To what extent, Pink1 deficiency enhanced neurological deficits and inflammatory responses after ICH can be attributed to resident microglia is an interesting research field.
In this study, the relative expression of MCP-1, one vital chemokine to regulate infiltration and migration of macrophages [21], is also significantly altered upon Pink1 deficiency.In a murine model of polymicrobial infection, Pink1-deficient macrophage shows a pronounced increase in Δψm, leading to the metabolic rewiring in macrophage activation [22].Mechanically, Pink1 improves retinoic-acid-inducible gene I-like receptors (RLRs)-triggered type I interferon production in macrophages by inhibiting TNF receptor-associated factor degradation [23].Some limitations should be acknowledged here.Although our previous analysis demonstrates that after ICH induction, down-regulated PINK1 expression mainly happened in microglia instead of astrocytes, oligodendrocytes, and neurons [24].Condition or microglia-specific knockout mice should be utilized to decipher the specific role of Pink1 in microglia in this study.On the other hand, only the relative expression of pro-and anti-inflammatory associated molecules is detected in the ipsilateral injury hemispheres, and cellular phenotype analysis should be performed in the future.Sex dimorphism has been demonstrated to affect experimental intracerebral hemorrhage, and only male mice are chosen in this study.The potential function of Pink1 deficiency on female ICH mice should also be investigated in the future.Last, the detailed mechanisms of how Pink1 deficiency impact ICH remain unclear.For example, it is not clear through what types of cells Pink1 functions during ICH.
Our study testifies that Pink1 deficiency can enhance ICH-caused neurological deficits and inflammatory responses.

Fig. 1 .
Fig. 1.Down-regulated Pink1 expression after intracerebral hemorrhage induction.The relative mRNA (A) and protein (B and C) levels of Pink1 were detected with qRT-PCR and Western blotting in ICH mice ipsilateral injury hemispheres.The time point of Pre-surgery was used as control.Image J was utilized for quantitative analysis, and expression was normalized to control (the time point of Pre-).The data was shown with mean AE SD. n ¼ 8 for each time point.*p < 0.05, **p < 0.01, ***p < 0.001 compared to control.One-way ANOVA followed Dunn's multiple comparisons test.

Fig. 4 .
Fig. 4. Pink1 deficiency enhanced brain edema and neurological deficits induced by ICH.Mouse neurological deficit scores (mNSS) were measured pre, 1, 3, 7, and 14 days post-ICH (A).n ¼ 10 for each group.Brain water content in the ipsilateral injury hemispheres was detected three days post-ICH (B).n ¼ 6 for each group.The data was shown with mean AE SD. #p < 0.05, ###p < 0.001 from Two-way ANOVA followed Tukey's multiple comparisons test.

Fig. 5 .
Fig. 5. Pink1 deficiency enhanced anxiety-like behavior induced by intracerebral hemorrhage.Pink deficiency increased the immobility time (A), as well as decreased the number of center crosses (B), the total distance traveled (C), and the velocity (D).n ¼ 10 for each group.The data was shown with mean AE SD. #p < 0.05, ##p < 0.01, ###p < 0.001 from Two-way ANOVA followed Tukey's multiple comparisons test.