Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: Primers used for quantification of mRNA expression in brain by RT-qPCR.

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Expressing, Sequencing

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) CB1R and (B.) CB2R mRNA were assessed in brain tissue by RT-qPCR and normalized to 18S expression. Data are expressed as fold change with respect to sham and revealed a selective 15-fold increase in CB2R expression. (C.) Histological analysis revealed increased CB2R+ immunoreactivity in the injured cortex (Scale-50µm), as compared to sham-operated brains. (D.) CB2R+ cells were quantified as mean fluorescent intensity and normalized to sham. (E.) Bone marrow chimera mice were generated whereby myeloid cells expressed red fluorescent protein (RFP). CB2R was expressed in 52.7% of RFP+CD11b+CD45HI infiltrating myeloid cells in the brain after TBI whereas 17.5% of RFP+ cells expressed CB2R in sham brains, as assessed by flow cytometry. (F) Quantification of RFP+CD11b+CD45HICB2R+ shown in panel (E.). Groups were compared as Student’s t-test. Data were represented as mean ± SEM (n=6; **p<0.01; ***p<0.001 vs. sham).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Quantitative RT-PCR, Expressing, Generated, Flow Cytometry

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) Schematic of macrophage (MΦ) adoptive transfer in recipient mice. CFSE-labeled CD11b+, CD68+, F4/80+ macrophages were intravenously administered through the tail vein immediately after sham/TBI. Brains were harvested for immunofluorescent staining at 72h post-injury. (B.) The increased infiltration of CFSE+ macrophages temporally and spatially paralleled increased CB2R+ expression after TBI (Scale-50µm). (C.) CB2R+ (red) and CFSE-labeled macrophages (green) were quantified as mean fluorescent intensity. Data were represented as mean ± SEM (n=6) and analyzed using Student’s t-test (***p<0.001 vs. sham).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Adoptive Transfer Assay, Labeling, Staining, Expressing

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) Representative flow cytometry dot plots showing CD68+ CD11b+ CB2R+ myeloid cells were sorted after sham/TBI and analyzed for microglia (CD45Low) and infiltrating macrophages (CD45High) populations. A TBI-induced increase in infiltrating macrophages was significantly reduced by treatment with GP1a (1 and 3 mg/kg), but not after treatment with the CB2R antagonist, AM360 (5 mg/kg). Interestingly, microglia did not show any significant alterations in numbers at day 3 post sham/TBI with or without treatment. Gating strategy has been shown in Fig 3A. Quantification of (B.) microglia and (C.) infiltrating macrophages are shown here. Data were represented as mean ± SEM and groups were compared by One-way ANOVA followed by Newman-Keuls multiple comparison. (n=6–7/group; *p<0.05; **p<0.01).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Flow Cytometry

Journal: Brain, behavior, and immunity

Article Title: Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization

doi: 10.1016/j.bbi.2017.10.021

Figure Lengend Snippet: (A.) Representative flow cytometry dot plots showing CB2R+ CD11b+ myeloid cells were sorted after sham/TBI and analyzed for M1 (CD68+ CD206− TNFα+) and M2 (CD68+ CD206+ IL10+) polarization. A TBI-induced increase in M1 polarization was significantly reduced by treatment with 3 mg/kg GP1a, but not after treatment with the CB2R antagonist, AM360 (5 mg/kg). Dot plots for GP1a treatments (1 mg/kg b.wt and 5 mg/kg b.wt.) are not shown here. Quantification of (B.) M2 macrophages (C.) M1 macrophages, and (D.) the ratio of M2:M1 macrophages after treatment. Data were represented as mean ± SEM and groups were compared by One-way ANOVA followed by Newman-Keuls multiple comparison. (n=6/group; *p<0.05; **p<0.01; ***p<0.001).

Article Snippet: Following an antigen retrieval step, deparaffinized sections were incubated with rabbit anti-CB2R antibody (1:200; Bioss#bs-2377R) overnight at 4°C followed by incubation with a fluorescently labeled secondary antibody (1:500; Jackson Laboratories, Burlingame, CA) for 1 hour.

Techniques: Flow Cytometry