Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: Study design. a Mice were randomly assigned to three groups: naive, sham, and surgery. Mice were sacrificed for tissues harvesting at 6 h, 1 day, and 3 days after surgery or sham. b Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + C3aR antagonist (C3aRa). C3aRa or vehicle was given 1 h prior to surgery or sham. Mice were sacrificed for tissues harvesting at 6 h and 1 day after surgery or sham. c Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + C3aR antagonist (C3aRa). 30 min after C3aRa or vehicle administration, mice were subjected to the training session for trace fear conditioning. Mice underwent surgery or sham 30 min after training. At 3 days after surgery or sham, context test was performed. d Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + recombinant mouse C3a (rmC3a). Mice were given intranasal rmC3a or vehicle at 24 h and 48 h after surgery or sham procedure. Mice were sacrificed for tissues harvesting at 3 days. e Naïve mice were randomly assigned to vehicle or rmC3a treatment; at 6 h after intranasal rmC3a or vehicle administration, mice were sacrificed for tissue harvesting. f Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + rmC3a. 30 min after rmC3a or vehicle administration, mice were trained for trace fear conditioning. Mice underwent surgery or sham 30 min after training. The context test was performed at 1 day after surgery or sham procedure

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques: Recombinant

Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: Orthopedic surgery induces complement activation in the hippocampus. a C3 was assayed by ELISA in hippocampal homogenates of naïve and surgery mice (one-way analysis of variance followed by Student-Newman-Keuls test; n = 5). b Representative confocal images of C3 (green) and astrocytic marker GFAP (red) immunostaining in the hippocampus of naïve, surgery, and sham mice on postoperative day 1. c Quantification of C3 occupancy in GFAP + astrocytes (one-way analysis of variance followed by Tukey post hoc test; n = 5). d Representative confocal images of C3 (green) and microglial marker IBA1 (red) double immunostaining in the hippocampus 1 day after surgery. e Representative confocal images of C3aR (green) and IBA1 (red) labeling in the hippocampus of naïve, surgery, and sham mice at 1 day. f Quantification of C3aR occupancy in IBA1 + microglia, normalized to the level in the naïve group (one-way analysis of variance followed by Tukey post hoc test; n = 5). Scale bar = 30 μm ( b , d , and e ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Marker, Immunostaining, Double Immunostaining, Labeling

Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: C3aR blockade ameliorates surgery-induced neuroinflammation. Mice were randomly assigned to three groups ( n = 5/group): sham + vehicle, surgery + vehicle, and surgery + C3aR antagonist (C3aRa). ELISA was performed to assay proinflammatory cytokines IL-1β ( a ) and IL-6 ( b ) in hippocampal homogenates at 6 h after surgery or sham procedure. c Representative confocal images of IBA1 (red) labeling in the hippocampus at 1 day after surgery or sham procedure. d Quantification of percentage of IBA1 + area. e Double immunostaining of adhesion molecular ICAM-1 (green) and endothelial marker CD31 (red) in the hippocampus. f Quantification of ICAM-1 by relative fluorescence intensity. g Representative images of anti-myeloperoxidase (MPO) (green) immunostaining; white arrows indicate MPO + cells. Nuclear counterstaining with DAPI (blue) ( c , g ). Scale bar = 100 μm ( c , e , and g ). Data analyses were performed using one-way analysis of variance followed by Tukey post hoc test; * p < 0.05, ** p < 0.01

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Double Immunostaining, Marker, Fluorescence, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: C3aR blockade reduces microglial phagocytic activity and synapse loss at 1 day after orthopedic surgery. Mice were randomly assigned to three groups ( n = 5/group): sham + vehicle, surgery + vehicle, and surgery + C3aR antagonist (C3aRa). a Representative confocal images of double immunostaining of CD68 (green) and IBA1 (red); scale bar = 30 μm. b Quantification of CD68 occupancy in IBA1 + microglia. Representative images from western blotting of presynaptic marker SYP ( c ) and postsynaptic marker PSD-95 ( e ). Quantification of SYP ( d ) and PSD-95 ( f ). Linear regression analyses of the relationship between microglia CD68 reactivity and synaptic marker SYP ( g ) or PSD-95 ( h ). Data analyses were performed using one-way analysis of variance followed by Tukey post hoc test ( b , d , f ) or regression analysis ( g , h ). ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques: Activity Assay, Double Immunostaining, Western Blot, Marker

Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: C3aR blockade attenuates BCSFB disruption in the choroid plexus after surgery. a Representative images of C3 labeling in the choroid plexus of naïve, surgery, and sham mice at 1 day after surgery or sham. b Quantification of C3 fluorescence intensity. c Representative images of IgG staining in the choroid plexus of 3 groups: sham + vehicle, surgery + vehicle, and surgery + C3aRa. d Quantification of IgG fluorescence intensity. e Representative images of ICAM-1 (green) and VCAM-1 (red) labelings in the choroid plexus. Quantification of ICAM-1 ( f ) and VCAM-1 ( g ) fluorescence intensity. Nuclear counterstaining with DAPI (blue) ( a , c , and e ). Scale bar = 100 μm ( a , c , and e ). Data analyses were performed using one-way analysis of variance followed by Tukey post hoc test ( b , d , f , and g ); ** p < 0.01, *** p < 0.001

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques: Labeling, Fluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: Hippocampal-dependent memory dysfunction after orthopedic surgery is ameliorated by C3aR blockade. Quantification of the percentage of freezing behavior during the context test on postoperative day 3. Data analyses were performed using one-way analysis of variance followed by Tukey post hoc test. ** p < 0.01, **** p < 0.0001

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Complement activation contributes to perioperative neurocognitive disorders in mice

doi: 10.1186/s12974-018-1292-4

Figure Lengend Snippet: Pharmacological activation of C3aR exacerbated PND-like pathology. ELISA was used to quantify hippocampal IL-1β ( a ) and IL-6 ( b ) on postoperative day 3 in three groups: sham + vehicle, surgery + vehicle, and surgery + rmC3a (one-way analysis of variance followed by Student-Newman-Keuls test; n = 5). c Representative images of MPO staining in the hippocampus of vehicle- and rmC3a-treated mice; white arrows indicate MPO + cells. d Representative images of IgG labeling in the choroid plexus of vehicle- and rmC3a-treated mice. Nuclear counterstaining with DAPI (blue) ( c , d ). e Quantification of IgG fluorescence intensity (unpaired Student’s t test; n = 5). f Quantification of the percentage of freezing time during the context on postoperative day 1 (one-way analysis of variance followed by Tukey post hoc test). Scale bar = 100 μm ( a and c ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: After blocking, the sections were incubated with primary antibodies against C3 (1:100, Abcam, #ab11862), C3aR (1:100, Hycult Biotech, #HM1123), ionized calcium-binding adapter molecule 1 (IBA1) (1:500, Wako, #019-19741), glial fibrillary acidic protein (GFAP) (1:1000, Dako, #Z0334), CD68 (1:200, Bio-Rad, #MCA1957), intercellular adhesion molecule-1 (ICAM-1) (1:200, R&D Systems, #AF796), vascular cell adhesion molecule-1 (VCAM-1) (1:100, Abcam, #134047), myeloperoxidase (MPO (1:100, Abcam, #ab9535), or mouse immunoglobulin G (IgG) (1:200, Invitrogen, #A-21203) overnight at 4 °C.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Fluorescence