Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury methods
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Materials and methods
Adult male and female C57BL/6 mice (22-27 g, aged 8-10 weeks) which were purchased from Slac Laboratory Co., Ltd. (Shanghai, China) were used for this study. Notably, male mice were used in this study except that 14 female mice were tested to compare the effect of sex differences on Mer expression and neurofunctional outcomes after TBI. All mice were housed in filter-top cages and fed a standard diet, with a 12-h light/dark cycle. Free access to food and water as well as controlled temperature and humidity were provided. All animal experiments were performed according to the Institutional Animal Care and Use Committee of Zhejiang University. The procedures were conducted according to the National Institutes of Health's Guide for the Care and the Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. All effor...
TBI model
TBI was induced in C57BL/6 mice using a CCI model (Suppl. Fig. A-C), as described previously [ ]. Briefly, mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and placed in the stereotaxic frame. A 4-mm-diameter craniotomy was performed using a portable drill over the right parietal cortex between bregma and lambda, 2 mm lateral to the midline. The dura mater was kept intact over the cortex. The CCI was performed perpendicular to the brain surface using a PinPoint™ Precision Cortical Impactor (Cary, NC, USA) with a 3-mm-diameter impact tip. The impact velocity is 3 m/s, the impact duration is 150 ms, and the impact depth is 2 mm. After TBI, the bone flap was immediately replaced and sealed, and the scalp was sutured closed. The animal's core body temperature was maintained at 37 ± 0.5 °C wi...
Intracerebroventricular (i.c.v.) injection
Previous studies demonstrated that i.c.v. delivery of siRNA can efficiently silence target gene expression in the brain with a range of 50-80% [ - ]. The i.c.v. administration of siRNA was performed as previously described [ ] (Suppl. Fig. D-F). A 1-mm-diameter burr hole was drilled into the right side of the skull (1.0 mm lateral and - 0.25 mm anterior-posterior (AP) to bregma). Mer siRNA or scrambled siRNA (500 pmol, Thermo Fisher Scientific) mixed with the transfection reagent (Engreen Biosystem Co., Ltd.), for a total volume of 2 µl, was delivered into the ipsilateral ventricle (1.0 mm lateral and - 0.25 mm AP to bregma, 2.5 mm dorsoventral (DV) below the skull). The injection was performed at a rate of 0.5 µl/min, then the needle stayed in the brain for another 5 min after injection to avert l...
Neurobehavioral function assessment
The mNSS score was assessed as previously reported [ ]. It includes sensory, motor, balance, and reflex tests. The neurological function was graded on a scale of 0-18 (normal score, 0; maximal deficit score, 18) and recorded before TBI, as well as at 1, 3, and 7 days after TBI.
Contusion volume assessment
To measure the contusion volume in the ipsilateral cortex 72 h after TBI, cresyl violet-stained sections were digitized and analyzed by using ImageJ (National Institutes of Health, Bethesda, MD, USA) as previously reported [ ]. The volume was computed by adding the injury areas and multiplying with the inter-slice distance (500 µm). Hemispheric tissue loss was expressed as a percentage that was calculated by the use of the following formulae: [(contralateral hemispheric volume - ipsilateral hemispheric volume)/(contralateral hemispheric volume) × 100%].
Western blot
Western blot was performed as previously described [ ]. Briefly, tissue samples from the ipsilateral cortex of sham/TBI mouse brains were homogenized in RIPA buffer (50 mM Tris-HCl at pH 7.4,150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) with protease and phosphatase inhibitors, followed by denaturation at 95 °C for 10 min. Then the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Next, the PVDF membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including anti-Mer antibody (1:1000, Abcam, ab184086), anti-p-Mer antibody (1:500, Abcam, ab192649), anti-STAT1 antibody (1:1000, CST, 14994), anti-p-STAT1 antibody (1: 1000, CST, 9167), anti-SOCS-1 (1:1000, Abcam, ab62584), anti-SOCS-3 (1:1...
Immunohistochemistry and confocal microscopy analysis
Mice were anesthetized and transcardially perfused with 0.1 mmol PBS and 4% paraformaldehyde (PFA) at 3 days post-TBI. Twenty-micrometer coronal cryosections were permeabilized and incubated in 5% Donkey Serum for 1 h for blocking. Then the brain tissues were incubated in primary antibody overnight at 4 °C. The primary antibody information is as follows: anti-Mer antibody (1:400, AF591, R&D Systems), anti-CD16/32 antibody (1:200, Abcam, ab25235), anti-CD206 antibody (1:400, Abcam ab64693), anti-Iba-1 antibody (1:500, Wako, 019-19741 or 1:500, Abcam, ab5076). After the incubation overnight, the cryosections were incubated with the secondary antibodies (1:500, Jackson Immunoresearch Laboratories) for 1 h at room temperature. After that, the sections were rinsed with PBS and covered with fluorescence mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (...
Expression patterns and cellular localization of Mer following TBI
Mer plays a critical role in regulating microglial/macrophage phenotypes and properties under physiological and pathological conditions [ ]. We first analyzed the levels of Mer protein and mRNA in the injured cortex at different time points after TBI (Suppl. Fig. A). As shown in Fig. a, b, both Mer protein and mRNA levels were significantly increased 12 h after TBI, peaked around day 3 and returned to the pre-injury levels around day 7. We further investigated the cellular localization and expression of Mer in microglia/macrophages in the ipsilateral cerebral cortex at 3 days after TBI, using double immunofluorescent staining of Mer and microglial/macrophage marker Iba-1. As demonstrated in Fig. c, e, Mer was abundantly expressed in the plasma membrane of microglia/macrophages in the sham group and substantially upregulated in the activated microglia/macrophages with phagocytoti...
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Adult male and female C57BL/6 mice (22-27 g, aged 8-10 weeks) which were purchased from Slac Laboratory Co., Ltd. (Shanghai, China) were used for this study. Notably, male mice were used in this study except that 14 female mice were tested to compare the effect of sex differences on Mer expression and neurofunctional outcomes after TBI. All mice were housed in filter-top cages and fed a standard diet, with a 12-h light/dark cycle. Free access to food and water as well as controlled temperature and humidity were provided. All animal experiments were performed according to the Institutional Animal Care and Use Committee of Zhejiang University. The procedures were conducted according to the National Institutes of Health's Guide for the Care and the Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. All efforts were made to minimize animal suffering and the number of animals sacrificed.
TBI was induced in C57BL/6 mice using a CCI model (Suppl. Fig. A-C), as described previously [ ]. Briefly, mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and placed in the stereotaxic frame. A 4-mm-diameter craniotomy was performed using a portable drill over the right parietal cortex between bregma and lambda, 2 mm lateral to the midline. The dura mater was kept intact over the cortex. The CCI was performed perpendicular to the brain surface using a PinPoint™ Precision Cortical Impactor (Cary, NC, USA) with a 3-mm-diameter impact tip. The impact velocity is 3 m/s, the impact duration is 150 ms, and the impact depth is 2 mm. After TBI, the bone flap was immediately replaced and sealed, and the scalp was sutured closed. The animal's core body temperature was maintained at 37 ± 0.5 °C with a thermostatically controlled heating pad during surgery. Sham animals were subjected to all aspects of the protocol (surgery, anesthesia, craniotomy, injection, and recovery) except for CCI. Recombinant PS (0.2 mg/kg) (9489-PS, R&D Systems) was administered via the tail vein at 1 h,...
Previous studies demonstrated that i.c.v. delivery of siRNA can efficiently silence target gene expression in the brain with a range of 50-80% [ - ]. The i.c.v. administration of siRNA was performed as previously described [ ] (Suppl. Fig. D-F). A 1-mm-diameter burr hole was drilled into the right side of the skull (1.0 mm lateral and - 0.25 mm anterior-posterior (AP) to bregma). Mer siRNA or scrambled siRNA (500 pmol, Thermo Fisher Scientific) mixed with the transfection reagent (Engreen Biosystem Co., Ltd.), for a total volume of 2 µl, was delivered into the ipsilateral ventricle (1.0 mm lateral and - 0.25 mm AP to bregma, 2.5 mm dorsoventral (DV) below the skull). The injection was performed at a rate of 0.5 µl/min, then the needle stayed in the brain for another 5 min after injection to avert leakage, and then the burr hole was sealed with bone wax, and the incision was closed with sutures. Mice were placed in individual recovery cages. The i.c.v. injection of siRNA was performed 1 day before and 10 min after TBI.
The mNSS score was assessed as previously reported [ ]. It includes sensory, motor, balance, and reflex tests. The neurological function was graded on a scale of 0-18 (normal score, 0; maximal deficit score, 18) and recorded before TBI, as well as at 1, 3, and 7 days after TBI.
To measure the contusion volume in the ipsilateral cortex 72 h after TBI, cresyl violet-stained sections were digitized and analyzed by using ImageJ (National Institutes of Health, Bethesda, MD, USA) as previously reported [ ]. The volume was computed by adding the injury areas and multiplying with the inter-slice distance (500 µm). Hemispheric tissue loss was expressed as a percentage that was calculated by the use of the following formulae: [(contralateral hemispheric volume - ipsilateral hemispheric volume)/(contralateral hemispheric volume) × 100%].
Western blot was performed as previously described [ ]. Briefly, tissue samples from the ipsilateral cortex of sham/TBI mouse brains were homogenized in RIPA buffer (50 mM Tris-HCl at pH 7.4,150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) with protease and phosphatase inhibitors, followed by denaturation at 95 °C for 10 min. Then the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Next, the PVDF membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including anti-Mer antibody (1:1000, Abcam, ab184086), anti-p-Mer antibody (1:500, Abcam, ab192649), anti-STAT1 antibody (1:1000, CST, 14994), anti-p-STAT1 antibody (1: 1000, CST, 9167), anti-SOCS-1 (1:1000, Abcam, ab62584), anti-SOCS-3 (1:1000, Abcam, ab16030), and anti-GAPDH (1:5000, Abcam, ab8245). After that, the PVDF membranes were disposed of with the relevant secondary antibodies (1:5000) for 1 h at room temperature and observed using the ECL kit chemiluminescence reagents (Millipore, Billerica, MA, USA). The signals of pr...
Mice were anesthetized and transcardially perfused with 0.1 mmol PBS and 4% paraformaldehyde (PFA) at 3 days post-TBI. Twenty-micrometer coronal cryosections were permeabilized and incubated in 5% Donkey Serum for 1 h for blocking. Then the brain tissues were incubated in primary antibody overnight at 4 °C. The primary antibody information is as follows: anti-Mer antibody (1:400, AF591, R&D Systems), anti-CD16/32 antibody (1:200, Abcam, ab25235), anti-CD206 antibody (1:400, Abcam ab64693), anti-Iba-1 antibody (1:500, Wako, 019-19741 or 1:500, Abcam, ab5076). After the incubation overnight, the cryosections were incubated with the secondary antibodies (1:500, Jackson Immunoresearch Laboratories) for 1 h at room temperature. After that, the sections were rinsed with PBS and covered with fluorescence mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, H-1200).
Mer plays a critical role in regulating microglial/macrophage phenotypes and properties under physiological and pathological conditions [ ]. We first analyzed the levels of Mer protein and mRNA in the injured cortex at different time points after TBI (Suppl. Fig. A). As shown in Fig. a, b, both Mer protein and mRNA levels were significantly increased 12 h after TBI, peaked around day 3 and returned to the pre-injury levels around day 7. We further investigated the cellular localization and expression of Mer in microglia/macrophages in the ipsilateral cerebral cortex at 3 days after TBI, using double immunofluorescent staining of Mer and microglial/macrophage marker Iba-1. As demonstrated in Fig. c, e, Mer was abundantly expressed in the plasma membrane of microglia/macrophages in the sham group and substantially upregulated in the activated microglia/macrophages with phagocytotic morphology (Fig. d, e) near the lesion at 3 days post-TBI. Fig. 2 Expression patterns and cellular localization of Mer following TBI. a Representative immunoblots and quantification showing the expression level of Mer protein in the injured cortex at 3 h, 12 h, 1 day, 3 days, and 7 day...
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"text": "Mer plays a critical role in regulating microglial/macrophage phenotypes and properties under physiological and pathological conditions [ ]. We first analyzed the levels of Mer protein and mRNA in the injured cortex at different time points after TBI (Suppl. Fig. A). As shown in Fig. a, b, both Mer protein and mRNA levels were significantly increased 12 h after TBI, peaked around day 3 and returned to the pre-injury levels around day 7. We further investigated the cellular localization and expression of Mer in microglia/macrophages in the ipsilateral cerebral cortex at 3 days after TBI, using double immunofluorescent staining of Mer and microglial/macrophage marker Iba-1. As demonstrated in Fig. c, e, Mer was abundantly expressed in the plasma membrane of microglia/macrophages in the sham group and substantially upregulated in the activated microglia/macrophages with phagocytoti..."
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