Animals and ICH Model
Adult male CD1 (8-week-old, 30-40 g, Charles River Laboratory, Wilmington, MA,USA) were used. Mice were housed in a room light of a 12-hour light/dark cycle and the temperature and humidity was controlled. All mice were free to access to water and food. ICH model was performed by striatal autologous blood injection as previously described 18.
Experimental Design
The following five separate experiments were performed.
Mice were randomized to experimental groups using a unique code linking to software-generated random numbers with 18.0 SPSS version. Investigators who conducted ICH surgeries and neurobehavioral tests were blinded to treatment assignments until the completion of data analysis.
Experiment 1
To examine the endogenous expressions of CCL17, Tregs marker Foxp3 and transforming growth factor-beta (TGF-β) after ICH, a total of 42 mice were randomly divided into 7 groups (n=6/group): sham, ICH-6h, ICH-12h, ICH-24h, ICH-72h,ICH-5d and ICH-7d for western blot. Additional 8 mice were used for immunofluorescence (IF) staining to localize Foxp3 in sham, ICH-24 h, ICH-72 h and ICH-7 d groups (n=2/group).
Experiment 2
To study the neuroprotective effect of endogenous Tregs following ICH, CD25-specific mAb was used to deplete Tregs at 48 h before ICH induction. Isotype control antibodies were used as control. This experiment was divided into two parts. The first part was to determine the efficacy of CD25-specific mAb on Tregs depletion after ICH. A total of 40 mice were randomly divided into 4 groups (n=10/group): sham + isotype control, sham + CD25-specific mAb, ICH + isotype control, ICH + CD25-specific mAb. Immunofluorescence (IF) staining and western blot were performed at 72 h post-ICH. The second part was to explore the protective effects of Tregs against ICH. 136 mice were randomly divided into the same 4 groups (n=34/group): sham + isotype control, sham + CD25-specific mAb, ICH + isotype control, ICH + CD25-specific mAb. Short-term neurobehavioral assessment (Corner Turn, Modified Garcia and Forelimb Placement Tests), hematoma volume and hemoglobin content were evaluated at 24 h, 72 h and 7 d following ICH. Brain water content was evaluated at 72 h post-ICH and immumohistochemical staining and western blot were used for assessing neuroinflammation at 72 h post-ICH.
Experiment 3
To determine the neuroprotective effect of rCCL17 was via upregulating peripheral Tregs recruitment into brain following ICH. This experiment was divided into two parts. The first part was to determine the effect of rCCL17 on peripheral Tregs recruitment after ICH. A total of 16 mice were randomly divided into 4 groups (n=4/group): sham + vehicle, sham + rCCL17 (30 μg/kg), ICH + vehicle, ICH + rCCL17 (30 μg/kg). Immumohistochemical and immunofluorescence staining were performed at 72 h post-ICH to evaluate the number of Tregs in ipsilateral brain hemisphere. Then, the second part was to explore whether neuroprotective effects of rCCL17 were depended on Tregs recruitment following ICH. A total of 72 mice were randomly divided into 4 groups (n=18/group): sham + isotype control, sham + CD25-specific mAb, ICH + isotype control, ICH + CD25-specific mAb. Neurobehavioral function, hematoma volume and hemoglobin content, brain water content, western blot were performed at 72 h post-ICH.
Experiment 4
To investigate the effects of Tregs recruitments on microglia/macrophage polarization after ICH, a total of 40 mice were randomized to 4 groups ((n=10/group)): ICH + isotype control, ICH + CD25-specific mAb, ICH + isotype control + rCCL17 (30 μg/kg), ICH + CD25-specific mAb + rCCL17 (30 μg/kg). Western blot and immunofluorescence staining were conducted to evaluate the microglia/macrophage polarization at 72 h following ICH.
Experiment 5
To investigate the mechanism of TGFβ/TGFβ1R/Smad2/3 pathway underlying the neuroprotective effects of Tregs in brain after ICH, a total of 50 mice were divided into 5 groups (n=10/group): sham, ICH + vehicle, ICH + rCCL17 (30 μg/kg), ICH + rCCL17 (30 μg/kg) + DMSO, ICH + rCCL17 (30 μg/kg) + SB431542. Western blot and immunofluorescence staining were performed at 72 h following ICH.
Drug Administration
At 1 h after ICH, rCCL17 (14013, LSBio, WA) at dose of 30 ug/kg was delivered intranasally (i.n.) as previously reported 18. SB431542 (ab120163, Abcam, USA) (1 μM solution, 100 μl/animal) was administrated intraperitoneally (i.p.) 1 h before the ICH induction. A total of 300mg of CD25-specific mAb (10200, biolegend, CA) or isotype control antiboy ((401602, biolegend, CA) was administered intraventricularly (i.c.v) route 48 h before the ICH induction as previously reported 19.
Short-term Neurobehavioral Assessment
The modified Garcia test with a twenty-one scoring system, corner turn test and the forelimb placement test (a positive percentage of total of 10 trials) were evaluated at 24 h, 72 h and 7 d after ICH as previously published from our lab20.
Brain Water Content Measurement
Brain water content was measured using the wet/dry method as previously reported 21. In brief, the brains were divided into 5 parts: left basal ganglia, left cortex, right basal ganglia, right cortex and cerebellum. Each part was measured immediately as the wet weight and then measured again as the dry weight after 48 h incubation in 100°C oven. Brain water content was calculated using the following formula: (Wet Weight−Dry Weight)/Wet Weight*100% to calculate.
Measurement of Hematoma Volume and Hemoglobin Content
Spectrophotometric measurement of hematoma volume and Drabkin’s reagent measurement of hemoglobin content were accessed at 24 h, 72 h and 7 d following the ICH induction as previously reported 19.
Western Blot
Western blot was performed as previously described 22. In brief, mice were perfused with cold PBS by transcardially. The right ipsilateral hemispheres were immediately harvested and mixed with RIPA. Supernatants were collected after centrifuged at 14 000g for 30 minutes (4°C). A total of 4 μL protein samples were loaded onto the SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated with the primary antibodies against CCL17 (1:1000, ab182793, Abcam, USA), Foxp3 (1:1000, ab182793, Abcam, USA), TGF-β (1:1000, GTX103322, Gene Tex), IL-1β (1:1000, ab9722, Abcam, USA), TNF-α (1:1000, ab6671, Abcam, USA), CD68 (1:1000, sc-97778, Santa Cruz Biotechnology, USA), CD206 (1:1000, sc-70585, Santa Cruz Biotechnology, USA), Smad2/3 (1:1000, Santa Cruz Biotechnology, USA) overnight at 4°C. The membranes were incubated with secondary antibodies at the second day and β-actin was used as a control. The relative density of the bands was analyzed with Image J software.
Immunofluorescence staining
Immunofluorescence staining was conducted at 72 h after ICH as previously described 23. Briefly, formalin fixed, frozen, brain samples were cut into 10 mm-thick sections. The sections were incubated with primary antibodies against Foxp3 (1:100, ab182793, Abcam, USA), CD68 (1:100, sc-97778, Santa Cruz Biotechnology, USA), CD206 (1:100, sc-70585, Santa Cruz Biotechnology, USA) over-night at 4°C. On the second day, the slices were incubated with appropriate secondary antibodies for 2 h at room temperature. After adding DAPI (4’, 6-diamidino-2-phe-nylindole) the slides were cover slipped. The staining were observed and photographed using a DMi8 fluorescent microscope under a 400 × fold field.
Immunohistochemistry
Immunohistochemistry was performed as described previously 24.Briefly, the mice were euthanized at 72 h after ICH. Brain samples were fixed in formalin, gradient dehydrated in 30% sucrose, and embed in OTC. The sliced brain samples were rinsed and blocked with 5% donkey serum. Myeloperoxidase (MPO), CD3, and Foxp3 immunochemistry staining was conducted using MPO antibody (1:200, ab208670, Abcam, USA), CD3 (1:200, sc-78588, Santa Cruz Biotechnology, USA), and Foxp3 antibody (1:100, ab182793, Abcam, USA). The staining was observed and photographed using a DMi8 fluorescent microscope under a 400 × fold field.
Statistical Analysis
Results were expressed as mean ± SD. The statistical analyses were performed using GraphPad Prism 7 (La Jolla, CA, USA). One-way ANOVA following by T-ukey post hoc test was used to evaluate quantitative western blot, brain water content and cell counting data. Two-way ANOVA following by T-ukey post hoc test was used to compare behavior data, hematoma volume and hemoglobin content. Statistical significance was considered as if P values were < 0.05.