Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Involvement of HMGB1-targeted receptors in the CIPN caused by bortezomib. Bortezomib at 0.4 mg/kg or vehicle was administered i.p. on days 0, 2, 5, 7, 9, and 12. ( A , B ) Preventive ( A ) or therapeutic ( B ) effect of FPS-ZM1, TAK-242, and AMD3100, antagonists of RAGE, TLR4, and CXCR4, respectively. The mice received repeated i.p. administration of FPS-ZM1 at 1 mg/kg, TAK-242 at 3 mg/kg, AMD3100 at 8 mg/kg, or vehicle, 30 min before each dose of bortezomib ( A ), or single i.p. administration of each of them on day 14 ( B ). ( C , D ) Protein levels of RAGE, TLR4, and CXCR4 in the dorsal root ganglion ( C ) and sciatic nerves ( D ) on day 14 after the onset of bortezomib treatment. ( E , F ) Lack of preventive ( E ) and therapeutic ( F ) effects of TH1020, a TLR5 antagonist. The mice received repeated ( E ) or single ( F ) i.p. administration of TH1020 at 1 mg/kg or vehicle, in mice treated with bortezomib according to the above-mentioned schedules. Data show the mean with S.E.M for 5–6 ( A , B , F ), 5–7 ( C ), 6–7 ( D ), or 6 ( E ) mice. V, vehicle; BTZ, bortezomib; DRG, dorsal root ganglion; * p < 0.05, ** p < 0.01 vs. V ( C ) or V in V-treated mice ( A , B , E , F ). † p < 0.05, †† p < 0.01 vs. V in BTZ-treated mice.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques:

Journal: Cells

Article Title: Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice

doi: 10.3390/cells10102550

Figure Lengend Snippet: Scheme for bortezomib-induced caspase-dependent HMGB1 release from macrophages and CIPN development, in contrast to caspase-independent mechanisms for paclitaxel. ( A ) Inhibition of proteasome by bortezomib causes caspase-dependent apoptosis of macrophages followed by the release of HMGB1, which in turn causes neuronal sensitization via activation of RAGE and acceleration of CXCL12/CXCR4 signals, leading to CIPN. ( B ) Paclitaxel causes HMGB1 release from macrophages through activation of the ROS/p38MAPK/NF-κB pathway , independently of caspase (see D), and the extracellular HMGB1 develops CIPN in a manner dependent on RAGE and CXCR4 , as shown in the CIPN caused by bortezomib.

Article Snippet: Primary antibodies were: an anti-HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, UK) (1: 5000 dilution), anti-RAGE rabbit polyclonal antibody (Abcam) (1:1000 dilution), anti-TLR4 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:10000 dilution), anti-CXCR4 rabbit polyclonal antibody (Novus Biologicals, Littleton, CO, USA) (1:1000 dilution), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) (1:5000 dilution).

Techniques: Inhibition, Activation Assay

Journal: Nature communications

Article Title: Neural progenitor cells orchestrate microglia migration and positioning into the developing cortex.

doi: 10.1038/ncomms6611

Figure Lengend Snippet: Figure 1 | Microglia distribution into the cerebral cortex overlaps Cxcl12 þ cortical domains. (a) Low magnification of the cerebral cortex at E10.5. High magnification (arrow) is shown in b (n ¼ 3). Labelling for Iba1 and CD45 in the cortical wall shows double-positive cells in the cortex and in the meninges (arrows indicate Iba1/CD45 þ cells). (c,d) Absolute numbers and cell densities of Iba1 þ cells were calculated by stereology at E14.5 and E18.5, and means±s.e.m. (pallium indicates the cerebral cortex and the hippocampus, while the sub pallium indicates basal ganglionic eminences and the septum, n ¼ 6 for each group). (e) Representative E16.5 cortical section probed for Cxcl12, boxed area indicates the VZ/SVZ of the lateral cerebral cortex. (f,g) High magnification sections labelled for Tbr2 and Iba1 (n ¼ 5). (h) Cxcl12 þ and Iba1 þ cell distribution at the corticostriatal boundary and into the Dentate Gyrus at E16.5 (n ¼ 5). (i) Representative RT–PCR for CxcR7, CxcR4 and the housekeeping gene H3 performed on P2 brain-derived primary microglia cell culture (primary MG) and on E15.5-sorted CD45low/CD11b þ cells (sorted MG; n ¼ 3 independent experiments). (j) Representative plot of CxcR4 þ cells, gated on CD45 þCD11b þLy6c cells from E15.5 brains (n ¼ 3 independent experiments). (k) Double labelling for Iba1 and CxcR4 in the cortical wall of E15.5 brain. Scale bar represents 100 mm. A two-tailed t-test was used to assess statistical significance: **Po0.01, ***Po0.001.

Article Snippet: The following antibodies were used: rabbit a-CxcR4 (Novus) 1:100; rabbit a-Iba1 (Wako) 1:400; rabbit a-Tbr2 (Abcam) 1:500; chicken a-GFP (Abcam) 1:500; rabbit a-pH3 (Cell Signaling) 1:200; rat a-CD206 (BD) 1:50; rabbit a-Ki67 (Novocastra) 1:100; rabbit a-CaB (Swant) 1:500; rat a-CD31 (BD) 1:100; rat a-BrdU (Abcam) 1:500; mouse a-IdU (BD) 1:75; rat a-CD45 (BD) 1:100; rabbit a-Casp3a (BD) 1:400; mouse a-F4/80 (Abcam) 1:200; rabbit a-Blbp (Millipore) 1:700; and click-it EdU alexafluor 647 imaging reagent (Invitrogen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Cell Culture, Two Tailed Test

Journal: Nature communications

Article Title: Neural progenitor cells orchestrate microglia migration and positioning into the developing cortex.

doi: 10.1038/ncomms6611

Figure Lengend Snippet: Figure 2 | The overexpression of Cxcl12 recruits microglia into the VZ/SVZ. Confocal images of E16.5 cortices receiving GFP plasmids (a,c,f,h) or GFP/Cxcl12 plasmids (b,d,e,g,i). (a,b) Double labelling for Blbp, GFP. (c,d) Double labelling for Tbr2 and GFP. (e) Ectopic Cxcl12 expression in the VZ/SVZ of Cxcl12-electroporated brains (n ¼ 5 for each group). (f,g) Distribution of Casp3a in GFP and GFP/Cxcl12-electroporated brains. (h,i) Confocal images of GFP (green), EdU (violet) and Iba1(red) in electroporated embryos pulsed with EdU for 3 h before killing. (j) Iba1 þ cells (means±s.e.m.) and Iba1/XdU þ cells (percentages±s.e.m.) in boxed areas (k; n ¼ 5 brains for each group). (l) Chemotactic responses of primary microglia to increasing amount of Cxcl12 and its reversion by AMD3100 (n ¼ 3). (m,n) Iba1 detection in E13.5 coronal sections from brains injected either with PBS (m) or AMD3100 (n). Cell counts (mean±s.e.m) are provided in o, (n ¼ 3 brains for each group). (p,q) Iba1 þ cells in E14.5 coronal sections from CxcR4 þ / and from CxcR4 / brains and their means±s.e.m. in r (n ¼ 3 brains for each group). Scale bar represents 100 mm. A two-tailed t-test was used to assess statistical significance: *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: The following antibodies were used: rabbit a-CxcR4 (Novus) 1:100; rabbit a-Iba1 (Wako) 1:400; rabbit a-Tbr2 (Abcam) 1:500; chicken a-GFP (Abcam) 1:500; rabbit a-pH3 (Cell Signaling) 1:200; rat a-CD206 (BD) 1:50; rabbit a-Ki67 (Novocastra) 1:100; rabbit a-CaB (Swant) 1:500; rat a-CD31 (BD) 1:100; rat a-BrdU (Abcam) 1:500; mouse a-IdU (BD) 1:75; rat a-CD45 (BD) 1:100; rabbit a-Casp3a (BD) 1:400; mouse a-F4/80 (Abcam) 1:200; rabbit a-Blbp (Millipore) 1:700; and click-it EdU alexafluor 647 imaging reagent (Invitrogen).

Techniques: Over Expression, Expressing, Injection, Two Tailed Test

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: UPARANT is an effective antiangiogenic agent in a mouse model of rubeosis iridis

doi: 10.1007/s00109-019-01794-w

Figure Lengend Snippet: List of antibodies

Article Snippet: Anti-CXCR4 , Rabbit , 1:200 , WB , Bio-Techne Corp., Abingdon, UK , NB100-56437.

Techniques:

Journal: The American Journal of Pathology

Article Title: Multifaceted C-X-C Chemokine Receptor 4 (CXCR4) Inhibition Interferes with Anti–Vascular Endothelial Growth Factor Therapy–Induced Glioma Dissemination

doi: 10.1016/j.ajpath.2017.04.020

Figure Lengend Snippet: Expression of CXCR4, and effect of POL5551 on CXCR4 ligand binding, cell viability, and migration in glioma cells in vitro. A: Using the Tag-lite CXCR4 ligand binding assay, CXCR4-expressing HEK293 cells were incubated with increasing concentrations of POL5551 or AMD3100 in the presence of a fixed concentration of fluorescent SDF-1α; the normalized responses were plotted, and the half-maximal inhibitory concentration was calculated. B: CT-2A and GL261 cells were both incubated under normoxic (N) and hypoxic (H; 1% O2) conditions for up to 24 hours and for 16 hours, respectively, and the protein expression of CXCR4, HIF-1α, and β-actin was determined by Western blot analysis. C: CT-2A, GL261, and U87 glioma cells were incubated in quadruplicate with POL5551 (1 nmol/L to 1 μmol/L) for 16 hours, and cell viability was assessed by MTT assay. D and E: In a Boyden chamber assay, CT-2A and GL261 cells were incubated in triplicate with POL5551 (0 to 500 nmol/L) under normoxic and hypoxic conditions and allowed to migrate in the absence or presence of an SDF-1α gradient (50 nmol/L) for 16 hours, after which the number of migrated cells was counted. F: To assess whether cellular proliferation contributed to the observed changes, cells were allowed to migrate in the presence of 10 mmol/L hydroxyurea for 16 hours. The results were analyzed by analysis of variance. Data are expressed as means ± SEM (A and C–F). ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: Western blotting was performed using rabbit anti-murine CXCR4 (1:250 dilution, NB100-56437; Novus Biologicals, Littleton, CO), rabbit anti-murine hypoxia-inducible factor (HIF)-1α (1:1000 dilution, A300-286A; Bethyl Laboratories, Montgomery, TX), and rabbit anti-murine β-actin (1:5000 dilution, A2066; Sigma-Aldrich) antibodies.

Techniques: Expressing, Ligand Binding Assay, Migration, In Vitro, Incubation, Concentration Assay, Western Blot, MTT Assay, Boyden Chamber Assay

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of HE-stained longitudinal sections of rat sciatic nerves. Left images are intact state, and others are 12 h after crush injury. The images marked with *and ** are high-magnification views of boxed areas at the epineurium and parenchyma, respectively. Arrows indicate neutrophils that exhibited a typical segmented or lobed morphology of the nucleus. Neutrophils were present at the injury site and at the epineurium but not the parenchyma in the region of WD. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively. (B) Quantification of the spatiotemporal distribution of neutrophils after injury (n = 3). Accumulation and disappearance of neutrophils were completed within 1 d after injury. (C) Quantification of neutrophils at 12 h after injury. Injuries were made at proximal or distal points in sciatic nerves, and data were combined (n = 3). Neutrophils accumulated in the region within 15 mm proximal to the injury site. Error bars represent the SEM. (D) Representative immunofluorescence images of neutrophils (Ly6G and NE), type 1 collagen (Col1), and DAPI in a longitudinal section at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and parenchyma. The image marked with * is a high-magnification view of the boxed area. Arrows indicate neutrophils at the epineurium. Scale bars: 50 and 10 μm in the low- and high-magnification images, respectively. (E) Representative immunofluorescence images of macrophages (CD68) in a longitudinal section at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and parenchyma. The image marked with * is a high-magnification view of the boxed area. Arrows indicate macrophages at the epineurium. Macrophages accumulated primarily at the epineurium and not the parenchyma in the region of WD. Scale bars: 50 and 10 μm in the low- and high-magnification images, respectively.

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Staining, Immunofluorescence

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of NE, Ly6G, and DAPI at 20 mm distal to the injury site 12 h after injury of Cl-amidine treated and control subjects. Scale bar: 50 μm. (B) Representative images of CD68-immunolabeled macrophages at the parenchyma and epineurium at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. Inhibition of NET formation by Cl-amidine or DNase I promoted the accumulation of macrophages at the parenchyma but reduced their accumulation at the epineurium. Scale bar: 50 μm. (C) Quantification of the total density of macrophages at the parenchyma and epineurium at 20 mm distal to the injury site 12 h after injury. There was no significant difference of the density of macrophages in the region of Wallerian degeneration among the control, Cl-amidine, and DNase I. One-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3). (D) Representative images of CCL2 immunoreactivity at the parenchyma at 20 mm distal to the injury site 12 h after injury. Scale bar: 50 μm. (E) Quantification of CCL2 immunoreactivity at the parenchyma at 20 mm distal to the injury site 12 h after injury. No significant difference was detected among the control, Cl-amidine, and DNase I. One-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Control, Immunolabeling, Inhibition

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of triple immunolabeling of longitudinal sections with MIF, Ly6G, and DAPI. Left is intact nerve, and other images were acquired at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. The images marked with * is a high-magnification image of the boxed area. Arrows indicate neutrophils expressing MIF. Neutrophils at the epineurium expressed MIF. Scale bars: 10 μm in * images and 20 μm in all other images. (B) Schematic illustration of an experimental method to inhibit MIF at the epineurium in Wallerian degeneration. The nerve of the region of Wallerian degeneration was wrapped with collagen sheets containing iso-1 or control. (C) Representative images of IgG immunolabeling at 20 mm distal to the injury site. No IgG immunoreactivity was detected at the parenchyma in either group. Scale bar: 500 μm. (D) Representative images of immunolabeling with CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border of the epineurium and the parenchyma. Images marked with * are high-magnification images of boxed areas in the epineurium. Arrows indicate triple immunolabeled NETs. Treatment with iso-1 dramatically inhibited NET formation. Scale bar: 10 μm. (E) Quantification of the NET formation detected by triple labeling with the CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Treatment with iso-1 significantly inhibited % of the area of triple immunoreactivity, indicating that iso-1 reduced NET formation. * P < 0.001; t test. Error bars represent the SEM (n = 3). (F) Representative images of CD68-labeled macrophages at the parenchyma at 20 mm distal to the injury site 12 h after injury. More macrophages were observed in animals treated with iso-1. Scale bar: 50 μm. (G) Quantification of macrophage density. The density of macrophages was significantly increased by iso-1 treatment. * P < 0.001; t test. Error bars represent the SEM (n = 3). (H) Representative images of HE-stained epineurium at 20 mm from the injury site. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 20 μm. (I) Quantification of the density of neutrophils accumulation at the epineurium. Treatment with iso-1 significantly increased the density of neutrophils at the epineurium compared with the control. * P < 0.05; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling, Expressing, Control, Labeling, Staining

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of sections triple-immunolabeled with MIF, Ly6G, and DAPI at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. Anti-PMN antibody administration reduced the expression of MIF at the epineurium. Scale bar: 50 μm. (B) Quantification of the MIF immunoreactive area at 20 mm distal to the injury site 12 h after injury. The anti-PMN antibody treatment significantly inhibited the area of MIF immunoreactivity. * P < 0.0001; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling, Expressing

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of triple immunolabeling of CXCR4, Ly6G, and DAPI of longitudinal sections. Left is the intact nerve, and others are at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. * is a high-magnification image of the boxed area. Arrows indicate neutrophils expressing CXCR4. Scale bars: 10 μm. (B) Scheme of experimental procedures to inhibit CXCR4 at the epineurium in Wallerian degeneration. The nerve of the region of Wallerian degeneration was wrapped with collagen sheets containing CXCR4 inhibitor, AMD3100, or control. (C) Representative images of IgG immunolabeling at 20 mm distal to the injury site. No immunoreactivity of IgG was detected at the parenchyma of the two groups. Scale bar: 500 μm. (D) Representative images of immunolabeling with CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border of the epineurium and the parenchyma. * are high-magnification images of boxed areas in the epineurium. Arrows indicate triple immunolabeled NETs. Scale bar: 10 μm. (E) Quantification of the NET formation detected by triple labeling with the CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. The treatment with AMD3100 significantly inhibited % of the area of triple immunoreactivity, indicating that AMD3100 reduced NET formation. * P < 0.001; t test. Error bars represent the SEM (n = 3). (F) Representative images of CD68-labeled macrophages at the parenchyma at 20 mm distal to the injury site 12 h after injury. More macrophages were observed in subjects receiving AMD3100. Scale bar: 50 μm. (G) Quantification of the density of macrophages. The density of macrophages was significantly increased by the AMD3100 treatment. * P < 0.001; t test. Error bars represent the SEM (n = 3). (H) Representative images of HE staining at 20 mm from the injury site. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 20 μm. (I) Quantification of the density of neutrophils accumulated at the epineurium (n = 3). Treatment with AMD 3100 significantly increased the density of neutrophils at the epineurium compared with the control. * P < 0.05; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling, Expressing, Control, Labeling, Staining

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of HE-stained longitudinal sections of rat sciatic nerves. Left images are intact state, and others are 12 h after crush injury. The images marked with *and ** are high-magnification views of boxed areas at the epineurium and parenchyma, respectively. Arrows indicate neutrophils that exhibited a typical segmented or lobed morphology of the nucleus. Neutrophils were present at the injury site and at the epineurium but not the parenchyma in the region of WD. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively. (B) Quantification of the spatiotemporal distribution of neutrophils after injury (n = 3). Accumulation and disappearance of neutrophils were completed within 1 d after injury. (C) Quantification of neutrophils at 12 h after injury. Injuries were made at proximal or distal points in sciatic nerves, and data were combined (n = 3). Neutrophils accumulated in the region within 15 mm proximal to the injury site. Error bars represent the SEM. (D) Representative immunofluorescence images of neutrophils (Ly6G and NE), type 1 collagen (Col1), and DAPI in a longitudinal section at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and parenchyma. The image marked with * is a high-magnification view of the boxed area. Arrows indicate neutrophils at the epineurium. Scale bars: 50 and 10 μm in the low- and high-magnification images, respectively. (E) Representative immunofluorescence images of macrophages (CD68) in a longitudinal section at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and parenchyma. The image marked with * is a high-magnification view of the boxed area. Arrows indicate macrophages at the epineurium. Macrophages accumulated primarily at the epineurium and not the parenchyma in the region of WD. Scale bars: 50 and 10 μm in the low- and high-magnification images, respectively.

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Staining, Immunofluorescence

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative immunofluorescence images of macrophages (CD68) in a longitudinal section at 20 mm distal to the injury site at 1, 6, 12 h, 1, 3, and 7 d after injury. Dashed lines indicate the border between the epineurium and parenchyma. The image marked with * is a high-magnification image of the boxed area. Macrophages accumulated only at the epineurium by 1 d after injury and later at the parenchyma in the Wallerian degeneration region. Scale bars: 25 and 5 μm in low- and high-magnification images, respectively. (B) Quantification of the spatiotemporal distribution of macrophages after injury (n = 3). The number of macrophages increases as a function of time after injury.

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunofluorescence

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of immunofluorescent labeling of IgG in longitudinal sections at intact nerve 12 h, 3, and 7 d after injury. Images of Wallerian degeneration (WD) sites were taken 20 mm from the injury site. Arrowheads indicate injury sites. IgG immunoreactivity was always detected at the epineurium (regardless of whether it was intact or damaged) at the injury site and at the parenchyma of WD 7 d after injury. Scale bar: 500 μm. (B) Schematic illustration of experimental methods to break down the BNB by mannitol and VEGF infusion. AA, abdominal aorta; CIA, common iliac artery. (C) Representative images of IgG immunolabeling at 20 mm from the injury site with or without forced BNB disruption. Apparent IgG immunoreactivity was detected at the parenchyma of forced BNB disruption. Scale bar: 500 μm. (D) Representative images of HE staining and NE immunolabeling at 20 mm from the injury site. Arrows indicate neutrophils. Neutrophils were observed at the parenchyma of forced BNB disruption. Scale bar: 10 μm. (E) Quantification of neutrophil accumulation at the parenchyma of the region of WD. BNB disruption significantly increased the number of neutrophils at the parenchyma. * P < 0.05; t test. Error bars represent the SEM (n = 3). (F) Representative images of CD68 immunolabeling. Dashed lines indicate the border between the parenchyma and the epineurium. The image marked with * is a high-magnification view of the boxed area. Arrows indicate macrophages. (G) Quantification of macrophage accumulation at the parenchyma of the region of WD. BNB disruption significantly increased the number of macrophages at the parenchyma. * P < 0.05; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Labeling, Immunolabeling, Disruption, Staining

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Time course of the experimental procedures to deplete neutrophils by anti-PMN antibody administration. (B) Quantification of neutrophils in the blood of each group: injury alone, Ctrl antibody, and anti-PMN antibody. Antibody administration depleted the increased neutrophils in blood circulation after peripheral nerve injury. * P < 0.05 versus injury alone and Ctrl antibody, ** P < 0.005 versus injury alone and Ctrl antibody, *** P < 0.05 versus injury alone. One-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3). (C) Representative HE images of the epineurium at 5 mm from the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. The epineurium became thinner and contained fewer neutrophils after anti-PMN antibody administration. Scale bar: 20 μm. (D) Quantification of neutrophil accumulation at the epineurium (n = 3). Anti-PMN antibody administration significantly reduced the number of neutrophils at the epineurium compared with the control antibody. * P < 0.05; t test. Error bars represent the SEM (n = 3). (E) Representative images of CD68-immunolabeled macrophages at the parenchyma 12 h after injury. Anti-PMN antibody administration promoted the accumulation of macrophages at the parenchyma. Scale bar: 50 μm. (F) Quantification of macrophage accumulation at the parenchyma 12 h after injury. Anti-PMN antibody administration significantly increased the macrophage density at the parenchyma. * P < 0.05, ** P < 0.01; t test. Error bars represent the SEM (n = 3). (G) Representative images of myelin debris immunolabeled with MBP at the parenchyma at 25 mm from the injury site 7 d after injury. Scale bar: 200 μm. (H) Quantification of MBP immunoreactivity at the parenchyma at 25 mm from the injury site 7 d after injury. Anti-PMN antibody administration significantly decreased the area of MBP immunoreactivity compared with control antibody administration. * P < 0.05; t test. Error bars represent the SEM (n = 3). (I) Representative images of regenerating axons immunolabeled with β3-tubulin 7 d after injury. Axon regeneration was enhanced in animals receiving the anti-PMN antibody compared with controls. Left is proximal. Scale bar: 200 μm. (J) Quantification of regenerating axons. Anti-PMN antibody administration significantly increased axon regeneration compared with control antibody administration * P < 0.05, ** P < 0.005; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Control, Immunolabeling

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of CD68-immunolabeled macrophages at the parenchyma and epineurium 12 h after injury. Anti-PMN antibody administration promoted the accumulation of macrophages at the parenchyma but reduced their accumulation in the epineurium. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 50 μm. (B) Quantification of the total density of macrophages at the parenchyma and epineurium at 20 mm distal to the injury site 12 h after injury. There was no significant difference of the density of macrophages in the region of Wallerian degeneration between the anti-PMN antibody and the control antibody administrations. t test. Error bars represent the SEM (n = 3). (C) Representative images of CCL2 immunoreactivity at the parenchyma at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 50 μm. (D) Quantification of CCL2 immunoreactivity at the parenchyma at 20 mm distal to the injury site 12 h after injury. No significant difference was detected between the anti-PMN antibody and the control antibody administrations. t test. Error bars represent the SEM (n = 3). (E) Representative images of CD68-immunolabeled macrophages at the parenchyma 7 d after injury. The anti-PMN antibody administration promoted the accumulation of macrophages at the parenchyma. Scale bar: 50 μm. (F) Quantification of the density of macrophages at the parenchyma 7 d after injury. The anti-PMN antibody administration significantly increased the macrophage density at the parenchyma. ** P < 0.01; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling, Control

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Time course of the experimental approach to increase circulating neutrophils. GFP-expressing mouse neutrophils were infused into mice 11 h after peripheral nerve injury. (B) Quantification of neutrophils in the blood 12 h after injury. Neutrophil infusion significantly increased the number of circulating neutrophils. * P < 0.001; t test. Error bars represent the SEM (n = 3). (C) Representative images of axial sections immunolabeled with GFP, NE, and DAPI at 20 mm from the injury site 12 h after injury. Dashed lines indicate the border of the epineurium and the parenchyma. Images marked with * are high-magnification images of boxed areas. Arrows indicate neutrophils. GFP-expressing neutrophils were detected only at the epineurium and not at the parenchyma. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively. (D) Quantification of neutrophil density 12 h after injury. Neutrophil infusion significantly increased the neutrophil density. * P < 0.001, ** P < 0.05; t test. Error bars represent the SEM (n = 3). (E) Representative images of macrophages immunolabeled with CD68 at the parenchyma 7 d after injury. Scale bar: 50 μm. (F) Quantification of the density of accumulating macrophages. Infusion of neutrophils into the blood significantly decreased the number of accumulating macrophages 7 d after injury. * P < 0.05; t test. Error bars represent the SEM (n = 3). (G) Representative image of myelin debris immunolabeled with MBP in axial sections 7 d after injury. Insets are high-magnification images of boxed areas. Dashed lines indicate the border between the epineurium and the parenchyma. More myelin debris was detected in neutrophil-infused animals than in controls. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively. (H) Quantification of myelin debris. The percentage of area exhibiting MBP immunoreactivity was significantly greater in neutrophil-infused animals than in controls. * P < 0.05; t test. Error bars represent the SEM (n = 3). (I) Representative images of axons immunolabeled with β3-tubulin in axial sections 7 d after injury. More labeled axons were detected in neutrophil-infused animals than in controls. Scale bar: 100 μm. (J) Quantification of axons. Neutrophil-infused animals exhibited significantly fewer axons than controls. Neutrophil infusion reduced the number of regenerating axons. * P < 0.05; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Expressing, Immunolabeling, Labeling

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of time course of NETs in the region of Wallerian degeneration. Longitudinal rat sciatic-nerve sections triply immunolabeled with CitH3, MPO, and DAPI. The left image shows the intact state, and others were at 20 mm distal to the injury site from 1 h to 7 d after crush injury. Dashed lines indicate the border between the epineurium and the parenchyma. Triple immunoreactivity was detected in the epineurium from 6 h to 1 d after injury and the greatest at the time point of 12 h after injury. Scale bars: 20 μm. (B) Low- and high-magnification images of the section triple immunolabeled with CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. The image marked with * is a high-magnification image of the boxed area at the epineurium. Dashed lines indicate the border between the epineurium and the parenchyma. Arrows indicate the NETs. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively. (C) Low- and high-magnification images of the section triple immunolabeled with CitH3, CD68, and DAPI at 20 mm distal to the injury site 12 h after injury. The image marked with † is a high-magnification image of the boxed area at the epineurium. Dashed lines indicate the border between the epineurium and the parenchyma. Arrowheads indicate macrophages surrounded by NETs detected by CitH3 and DAPI. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively.

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Schematic illustration of an experimental method to inhibit NET formation at the epineurium in Wallerian degeneration. The nerve of the region of Wallerian degeneration was wrapped with collagen sheets containing the PAD4 inhibitor Cl-amidine, DNase I, or control. (B) Representative images of IgG immunolabeling at 20 mm distal to the injury site. No IgG immunoreactivity was detected at the parenchyma in any group, indicating no blood–nerve barrier disruption. Scale bar: 500 μm. (C) Representative images of immunolabeling with CitH3, MPO, and DAPI at 20 mm distal to the injury site. Dashed lines indicate the border of the epineurium and the parenchyma. Images marked with * are high-magnification views of the boxed areas at the epineurium. Arrows indicate neutrophils releasing NETs. Treatment with Cl-amidine or DNase I inhibited NET formation. Scale bars: 100 and 10 μm in low- and high-magnification images, respectively. (D) Quantification of the NET formation detected by triple immunolabeling with the CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Treatment with Cl-amidine or DNase I significantly inhibited NET formation. * P < 0.001; one-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3). (E) Representative images of CD68-labeled macrophages at the parenchyma at 20 mm distal to the injury site 12 h after injury. More macrophages were observed in animals receiving Cl-amidine or DNase I. Scale bar: 50 μm. (F) Quantification of macrophage density. Macrophage density was significantly increased by Cl-amidine or DNase I treatment. * P < 0.001; one-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3). (G) Representative images of HE-stained epineurium at 20 mm from the injury site. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 20 μm. (H) Quantification of the density of neutrophils accumulated at the epineurium. Treatment with Cl-amidine and DNaseⅠ significantly increased the density of neutrophils at the epineurium compared with the control. * P < 0.005, ** P < 0.05; one-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Control, Immunolabeling, Disruption, Labeling, Staining

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of NE, Ly6G, and DAPI at 20 mm distal to the injury site 12 h after injury of Cl-amidine treated and control subjects. Scale bar: 50 μm. (B) Representative images of CD68-immunolabeled macrophages at the parenchyma and epineurium at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. Inhibition of NET formation by Cl-amidine or DNase I promoted the accumulation of macrophages at the parenchyma but reduced their accumulation at the epineurium. Scale bar: 50 μm. (C) Quantification of the total density of macrophages at the parenchyma and epineurium at 20 mm distal to the injury site 12 h after injury. There was no significant difference of the density of macrophages in the region of Wallerian degeneration among the control, Cl-amidine, and DNase I. One-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3). (D) Representative images of CCL2 immunoreactivity at the parenchyma at 20 mm distal to the injury site 12 h after injury. Scale bar: 50 μm. (E) Quantification of CCL2 immunoreactivity at the parenchyma at 20 mm distal to the injury site 12 h after injury. No significant difference was detected among the control, Cl-amidine, and DNase I. One-way ANOVA with the Turkey-Kramer test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Control, Immunolabeling, Inhibition

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of triple immunolabeling of longitudinal sections with MIF, Ly6G, and DAPI. Left is intact nerve, and other images were acquired at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. The images marked with * is a high-magnification image of the boxed area. Arrows indicate neutrophils expressing MIF. Neutrophils at the epineurium expressed MIF. Scale bars: 10 μm in * images and 20 μm in all other images. (B) Schematic illustration of an experimental method to inhibit MIF at the epineurium in Wallerian degeneration. The nerve of the region of Wallerian degeneration was wrapped with collagen sheets containing iso-1 or control. (C) Representative images of IgG immunolabeling at 20 mm distal to the injury site. No IgG immunoreactivity was detected at the parenchyma in either group. Scale bar: 500 μm. (D) Representative images of immunolabeling with CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border of the epineurium and the parenchyma. Images marked with * are high-magnification images of boxed areas in the epineurium. Arrows indicate triple immunolabeled NETs. Treatment with iso-1 dramatically inhibited NET formation. Scale bar: 10 μm. (E) Quantification of the NET formation detected by triple labeling with the CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Treatment with iso-1 significantly inhibited % of the area of triple immunoreactivity, indicating that iso-1 reduced NET formation. * P < 0.001; t test. Error bars represent the SEM (n = 3). (F) Representative images of CD68-labeled macrophages at the parenchyma at 20 mm distal to the injury site 12 h after injury. More macrophages were observed in animals treated with iso-1. Scale bar: 50 μm. (G) Quantification of macrophage density. The density of macrophages was significantly increased by iso-1 treatment. * P < 0.001; t test. Error bars represent the SEM (n = 3). (H) Representative images of HE-stained epineurium at 20 mm from the injury site. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 20 μm. (I) Quantification of the density of neutrophils accumulation at the epineurium. Treatment with iso-1 significantly increased the density of neutrophils at the epineurium compared with the control. * P < 0.05; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling, Expressing, Control, Labeling, Staining

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Representative images of triple immunolabeling of CXCR4, Ly6G, and DAPI of longitudinal sections. Left is the intact nerve, and others are at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border between the epineurium and the parenchyma. * is a high-magnification image of the boxed area. Arrows indicate neutrophils expressing CXCR4. Scale bars: 10 μm. (B) Scheme of experimental procedures to inhibit CXCR4 at the epineurium in Wallerian degeneration. The nerve of the region of Wallerian degeneration was wrapped with collagen sheets containing CXCR4 inhibitor, AMD3100, or control. (C) Representative images of IgG immunolabeling at 20 mm distal to the injury site. No immunoreactivity of IgG was detected at the parenchyma of the two groups. Scale bar: 500 μm. (D) Representative images of immunolabeling with CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. Dashed lines indicate the border of the epineurium and the parenchyma. * are high-magnification images of boxed areas in the epineurium. Arrows indicate triple immunolabeled NETs. Scale bar: 10 μm. (E) Quantification of the NET formation detected by triple labeling with the CitH3, MPO, and DAPI at 20 mm distal to the injury site 12 h after injury. The treatment with AMD3100 significantly inhibited % of the area of triple immunoreactivity, indicating that AMD3100 reduced NET formation. * P < 0.001; t test. Error bars represent the SEM (n = 3). (F) Representative images of CD68-labeled macrophages at the parenchyma at 20 mm distal to the injury site 12 h after injury. More macrophages were observed in subjects receiving AMD3100. Scale bar: 50 μm. (G) Quantification of the density of macrophages. The density of macrophages was significantly increased by the AMD3100 treatment. * P < 0.001; t test. Error bars represent the SEM (n = 3). (H) Representative images of HE staining at 20 mm from the injury site. Dashed lines indicate the border between the epineurium and the parenchyma. Scale bar: 20 μm. (I) Quantification of the density of neutrophils accumulated at the epineurium (n = 3). Treatment with AMD 3100 significantly increased the density of neutrophils at the epineurium compared with the control. * P < 0.05; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Immunolabeling, Expressing, Control, Labeling, Staining

Journal: Life Science Alliance

Article Title: Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma

doi: 10.26508/lsa.202201399

Figure Lengend Snippet: (A) Time course of the experimental procedures to inhibit migration inhibitory factor at the epineurium in WD. The nerve of the region of WD was wrapped with collagen sheets containing iso-1 or control for 2 h, followed by perfusion a week later. (B) Representative images of CD68-immunolabeled macrophages at the parenchyma 7 d after injury. The iso-1 treatment promoted the accumulation of macrophages at the parenchyma. Scale bar: 50 μm. (C) Quantification of macrophage accumulation at the parenchyma 7 d after injury. The iso-1 treatment significantly increased the macrophage density at the parenchyma. * P < 0.005; t test. Error bars represent the SEM (n = 3). (D) Representative images of myelin debris immunolabeled with MBP at the parenchyma at 25 mm from injury site at 7 d after injury. The iso-1 treatment reduced myelin debris. Scale bar: 200 μm. (E) Quantification of MBP immunoreactivity at the parenchyma at 25 mm from injury site 7 d after injury. The iso-1 treatment significantly decreased MBP immunoreactivity area compared with the control. * P < 0.001; t test. Error bars represent the SEM (n = 3). (F) Representative images of axon regeneration immunolabeled with β3tubulin at 7 d after injury. Axons regenerated greater in iso-1 treatment subjects than in the control subjects. Left is proximal. Scale bar: 200 μm. (G) Quantification of regenerating axons. The iso-1 treatment significantly increased axon regeneration compared with the control. * P < 0.05, ** P < 0.005; t test. Error bars represent the SEM (n = 3).

Article Snippet: Primary antibodies were NE (1:200 rabbit from Abcam #ab68672) and Ly6G (1:200, rat from GeneTex #GTX40913) to detect neutrophils, CD68 (ED1; 1/500, mouse from Bio-Rad #MCA341R) to detect macrophages, biotin-conjugated donkey anti-rat IgG (1/500, rabbit from Jackson ImmunoResearch #712-065-150) to detect IgG, β3tublin (1/1,000, mouse from BioLegend #801202) or β3tublin (1/1,000, rabbit from Covance #PRB-435P) to detect axons, MBP (1/1,000, chicken from Aves lab #AB_2313550) to detect myelin, typeIcollagen (1/200, chicken from SouthernBiotech #1301-01), GFP (1/1,000, rabbit from Thermo Fisher Scientific #a6455), CCL2 (1/200, rabbit from Thermo Fisher Scientific #PA5-115555), citrullinated histone 3 (1/100, rabbit from Abcam #ab5103) and MPO (1/100, goat from R & D System #AF3667) to detect NETs ( ; ), CXCR4 (1/100, rabbit from Novus #NB100-56437), and MIF (1/200, rabbit from Thermo Fisher Scientific #PA527343).

Techniques: Migration, Control, Immunolabeling