Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke methods
Aim. Evidence-backed execution summary for Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke methods from Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
In-vivo multiphoton microscopy
reagent used in the protocol.
- Use
- To detect neutrophils, phycoerythrin (PE)-conjugated monoclonal Ly6G antibody (1A8 clone; 3 µg, 12-9668-80, eBioscience) was injected i.v. into mice. Blood vessels were visualized by i.v. injection of FITC-dextran (2,000,000 Da, Sigma-Aldrich, 0.1 mL of 10 mg/mL). Time-lapse images were...
Stroke induces NET formation
reagent used in the protocol.
- Use
- To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (F...
Disruption of NETs enhances vascular remodeling after stroke
reagent used in the protocol.
- Use
- We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrophils in the peri-infarct cortical areas (Supplementary Fig. ) but reduced H3Cit levels (Fig. ). Compared with the vehicle contro...
Disruption of NETs enhances vascular remodeling after stroke
reagent used in the protocol.
- Use
- Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and pe...
PAD4 regulates BBB permeability and neovascularization
reagent used in the protocol.
- Use
- Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-f...
PAD4 regulates BBB permeability and neovascularization
reagent used in the protocol.
- Use
- To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig....
NETs are responsible for STING-mediated vascular remodeling
reagent used in the protocol.
- Use
- The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly i...
Neutrophil depletion
reagent used in the protocol.
- Use
- Mice received an intraperitoneal (i.p.) injection of 100 µg monoclonal anti-mouse Ly6G (1A8 clone; specific for neutrophils, BE0075-1, BioXCell, NH) 24 h or 7 days after cerebral ischemia. The mice were then injected every second day for 3, 7, or 14 days until killing. Rat IgG2a isotype control was...
Methods
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6...
- Use
- Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6...
Methods
For cerebral infarction, six coronal sections (20 µm) were stained with hematoxylin and eosin. Infarct volume was measured using the NIH ImageJ 1.46r software and was presented as percentage of the contralateral hemisphere.
- Use
- For cerebral infarction, six coronal sections (20 µm) were stained with hematoxylin and eosin. Infarct volume was measured using the NIH ImageJ 1.46r software and was presented as percentage of the contralateral hemisphere.
In-vivo multiphoton microscopy
Mice were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide, and kept on a heating plate (37 ± 0.5 °C). After fixation in a custom-made head holder, a craniotomy was created over the right somatosensory cortex (centered 2.5 mm lateral and 1.5 mm pos...
- Use
- Mice were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide, and kept on a heating plate (37 ± 0.5 °C). After fixation in a custom-made head holder, a craniotomy was created over the right somatosensory cortex (centered 2.5 mm lateral and 1.5 mm pos...
In-vivo multiphoton microscopy
To analyze microvascular perfusion, a 0.1 mL bolus of 10 mg/mL FITC-dextran (2,000,000 Da, Sigma-Aldrich; St. Louis, MO) was injected i.v.,,. Z -stack images at 5 µm steps were acquired from 200 to 500 µm below the surface of the cortex. The area scanned was 500 ×...
- Use
- To analyze microvascular perfusion, a 0.1 mL bolus of 10 mg/mL FITC-dextran (2,000,000 Da, Sigma-Aldrich; St. Louis, MO) was injected i.v.,,. Z -stack images at 5 µm steps were acquired from 200 to 500 µm below the surface of the cortex. The area scanned was 500 ×...
Neutrophil depletion
Mice received an intraperitoneal (i.p.) injection of 100 µg monoclonal anti-mouse Ly6G (1A8 clone; specific for neutrophils, BE0075-1, BioXCell, NH) 24 h or 7 days after cerebral ischemia. The mice were then injected every second day for 3, 7, or 14 days until killing. Rat IgG2a isotype control was...
- Use
- Mice received an intraperitoneal (i.p.) injection of 100 µg monoclonal anti-mouse Ly6G (1A8 clone; specific for neutrophils, BE0075-1, BioXCell, NH) 24 h or 7 days after cerebral ischemia. The mice were then injected every second day for 3, 7, or 14 days until killing. Rat IgG2a isotype control was...
Blood counts
At 14 days after cerebral ischemia, mice were bled from the retro-orbital plexus under isoflurane anesthesia. Blood was collected into a EDTA-containing tube. The samples were analyzed using a SYSME X Hematology Analyzer (Sysme, Japan).
- Use
- At 14 days after cerebral ischemia, mice were bled from the retro-orbital plexus under isoflurane anesthesia. Blood was collected into a EDTA-containing tube. The samples were analyzed using a SYSME X Hematology Analyzer (Sysme, Japan).
Flow cytometry
Peripheral blood was subjected to red blood cell lysis buffer (155 mmol/L NH 4 Cl, 10 mmol/L KHCO 3, and 0.1 mmol/L Na 2 EDTA). Cells were washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and resuspended in rat anti-mouse CD16/32 Fc block (2.4G2 clone; 1:...
- Use
- Peripheral blood was subjected to red blood cell lysis buffer (155 mmol/L NH 4 Cl, 10 mmol/L KHCO 3, and 0.1 mmol/L Na 2 EDTA). Cells were washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and resuspended in rat anti-mouse CD16/32 Fc block (2.4G2 clone; 1:...
Cytospin NET analysis
Whole blood collected from the retro-orbital sinus was lysed with red blood cell lysis buffer. The cells were resuspended in 7.5% BSA in PBS and plated on slides using a Shandon Cytospin 4 (Thermo Scientific). Slides were fixed in 4% paraformaldehyde at 4 °C overnight and incubated with rabbit anti-H3Cit...
- Use
- Whole blood collected from the retro-orbital sinus was lysed with red blood cell lysis buffer. The cells were resuspended in 7.5% BSA in PBS and plated on slides using a Shandon Cytospin 4 (Thermo Scientific). Slides were fixed in 4% paraformaldehyde at 4 °C overnight and incubated with rabbit anti-H3Cit...
PAD4 regulates BBB permeability and neovascularization
Software used for acquisition, scoring, statistics, or reporting.
- Use
- To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig....
Neutrophil depletion
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Mice received an intraperitoneal (i.p.) injection of 100 µg monoclonal anti-mouse Ly6G (1A8 clone; specific for neutrophils, BE0075-1, BioXCell, NH) 24 h or 7 days after cerebral ischemia. The mice were then injected every second day for 3, 7, or 14 days until killing. Rat IgG2a isotype control was...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The data were analyzed using GraphPad Prism 7 software. All values are presented as mean ± SD. Multiple comparisons were analyzed by one-way analysis of variance followed by the Bonferroni multiple comparison test. When comparing two groups, unpaired Student's t -test or Mann-Whitney tes...
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Stroke induces NET formation
To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (Fig. ). In line with this, elevated levels of circulating DNA was found in the plasma from these mice (Fig. ). We then isolated neutrophils from the peripheral blood of these mice and incubated them with or without lipopolysaccharide (LPS) stimulation. We found that either unstimulated or LPS-stimulated neutrophils from ischemic mice showed a significant increase in H3Cit + neutrophils and NET formation (Fig. ), indicating that neutrophils from mice subjected to stroke are primed to undergo NETosis. Fig. 3 Neutrophils form NETs presenting in the brain after...
Stroke induces NET formation
As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed an increased amount of H3Cit that was most robust from 3-5 days (Fig. ), suggesting that NETs may play an important role during the delayed phases after stroke. Immunostaining revealed that the peri-infarct cortex was extensively labeled with H3Cit + cells at 3 days (Fig. ). To identify which type of cells expressed H3cit after stroke, double immunofluorescence with confocal microscopy was performed on brain sections. This analysis revealed that H3Cit was colocalized with Ly6G-positive neutrophils, F4/80-positive macrophages/microglial cells, Iba1-positive microglial cells, NeuN-positive neurons, and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. ). Importantly, 78.7% of...
Disruption of NETs enhances vascular remodeling after stroke
We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrophils in the peri-infarct cortical areas (Supplementary Fig. ) but reduced H3Cit levels (Fig. ). Compared with the vehicle controls, administration of DNase 1 significantly reduced BBB permeability (Fig. ) and extravascular IgG deposits (Fig. ), and enhanced both Pdgfr-β + and CD13 + pericyte coverage on brain microvessels (Fig. and Supplementary Fig. ). Vascular branches (Supplementary Fig. ), microvascular length (Fig. ), perfused capillary length (Fig. ), and tomato-lectin perfused vessels (Fig. ) were also increased in the brains of mice treated with DNase 1. Next, we investigated the importance of neutrophil NETs in vascular remodeling. Our re...
Disruption of NETs enhances vascular remodeling after stroke
Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and perfused cortical vessels (Fig. ) in anti-Ly6G antibody-treated mice compared with control IgG-treated mice. Treatment with DNase 1 starting at 7 days after stroke also attenuated BBB disruption (Fig. ), increased microvessels (Fig. ), and improved capillary perfusion (Fig. ) at 14 days. Fig. 5 Increased vascular remodeling by delayed inhibition of NET formation. a - d Representative confocal images ( a, c ) and quantitative analysis of IgG extravascular deposits ( b, d ) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and...
PAD4 regulates BBB permeability and neovascularization
Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-fold upregulation of PAD4 protein expression in these areas (Fig. ). To test the hypothesis that increased release of NETs, orchestrated by PAD4, participates in stroke recovery, we first studied the role of overexpression of PAD4 on vascular remodeling. Immunohistochemical analysis indicated extensive expression of recombinant adeno-PAD4-flag-infected cells in the cortex at 4 days after injection (Fig. ). Overexpression of PAD4 in the cortex was also confirmed by immunoblotting (Supplementary Fig. ). PAD4-flag was present in the neurons, neutrophils, macro...
PAD4 regulates BBB permeability and neovascularization
To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig. ). Thus, this approach allowed us to provide evidence that effects of PAD4 deficiency on long-term outcomes are not secondary to the reduced lesion size. PAD4 deficiency did not alter the number of infiltrating neutrophils in the ischemic brain tissue (Supplementary Fig. ), whereas the levels of H3Cit in the lysates from the ischemic cortex at 3 days were greatly reduced in PAD4 -/- mice and mice receiving Cl-amidine (Fig. and Supplementary Fig. ). Similarly, examination of the brain tissues revealed that PAD4 deficiency or Cl-amidine reduced...
NETs are responsible for STING-mediated vascular remodeling
The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly increased levels of STING (Fig. and Supplementary Fig. ) and robust induction of phosphorylated TANK-binding kinase 1 (pTBK1) and TBK1-dependent IFN regulatory factor 3 (IRF3) activation (Fig. and Supplementary Fig. ). Treatment with the PAD inhibitor Cl-amidine reduced the levels of stroke-induced STING-meditated signaling in the cortical areas compared with vehicle-treated mice (Fig. and Supplementary Fig. ). Neutrophil depletion also resulted in a significant reduction in the levels of STING and the STING downstream signaling molecules...
Methods
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used as controls. All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light-dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%. Focal cortical cerebral ischemia was induced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA),. Male mice weighing 23-26 g were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Afte...
Measurement outputs
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To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. Th...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed a...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrop...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extr...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. Th...; As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed a...; We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrop...; Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extr....
from paperStatistical comparison
To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. Th...; We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrop...; Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extr...; Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA ex...
from paperReporting output
Report representative outputs alongside summary comparisons for To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. Th..., As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed a..., We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrop..., Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extr....
inferred from protocolStructured statistical methods
To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. Th...; We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrop...; Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extr...; Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA ex...
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To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (Fig. ). In line with this, elevated levels of circulating DNA was found in the plasma from these mice (Fig. ). We then isolated neutrophils from the peripheral blood of these mice and incubated them with or without lipopolysaccharide (LPS) stimulation. We found that either unstimulated or LPS-stimulated neutrophils from ischemic mice showed a significant increase in H3Cit + neutrophils and NET formation (Fig. ), indicating that neutrophils from mice subjected to stroke are primed to undergo NETosis. Fig. 3 Neutrophils form NETs presenting in the brain after stroke. a Representative images of H3Cit (green) and Ly6G (red) double-positive cells in cytospins from sham-operated mice and ischemic mice at 3 days. DNA was visualized with Hoechst 33342 (blue). Bar = 30 µm. b, c Quantification of Ly6G-positive neutrophils in the total le...
As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed an increased amount of H3Cit that was most robust from 3-5 days (Fig. ), suggesting that NETs may play an important role during the delayed phases after stroke. Immunostaining revealed that the peri-infarct cortex was extensively labeled with H3Cit + cells at 3 days (Fig. ). To identify which type of cells expressed H3cit after stroke, double immunofluorescence with confocal microscopy was performed on brain sections. This analysis revealed that H3Cit was colocalized with Ly6G-positive neutrophils, F4/80-positive macrophages/microglial cells, Iba1-positive microglial cells, NeuN-positive neurons, and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. ). Importantly, 78.7% of the H3Cit-positive cells were Ly6G-positive neutrophils. H3Cit + neutrophils were observed inside the blood vessels and cerebral parenchyma (Fig. ). Hematoxylin and eosin staining clearly indicated that DNA fibers were present in these areas (Supplementary Fig. ). To confirm these obser...
We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrophils in the peri-infarct cortical areas (Supplementary Fig. ) but reduced H3Cit levels (Fig. ). Compared with the vehicle controls, administration of DNase 1 significantly reduced BBB permeability (Fig. ) and extravascular IgG deposits (Fig. ), and enhanced both Pdgfr-β + and CD13 + pericyte coverage on brain microvessels (Fig. and Supplementary Fig. ). Vascular branches (Supplementary Fig. ), microvascular length (Fig. ), perfused capillary length (Fig. ), and tomato-lectin perfused vessels (Fig. ) were also increased in the brains of mice treated with DNase 1. Next, we investigated the importance of neutrophil NETs in vascular remodeling. Our results showed that treatment with DNase 1 in combination with anti-Ly6G antibody did not further improve neovascularization and BBB leakage compared with mice treated with anti-Ly6G antibody alone (Fig. ). These data indicate that DNase 1 primarily digests NETs generated by neutrophils, and tha...
Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and perfused cortical vessels (Fig. ) in anti-Ly6G antibody-treated mice compared with control IgG-treated mice. Treatment with DNase 1 starting at 7 days after stroke also attenuated BBB disruption (Fig. ), increased microvessels (Fig. ), and improved capillary perfusion (Fig. ) at 14 days. Fig. 5 Increased vascular remodeling by delayed inhibition of NET formation. a - d Representative confocal images ( a, c ) and quantitative analysis of IgG extravascular deposits ( b, d ) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and treated with either anti-Ly6G antibody, control antibody, DNase 1, or vehicle starting at 7 days ( n = 6), unpaired two-tailed Student's t -test was applied with * P = 0.0392 ( b ), * P = 0.0384 ( d ). Bar = 10 µm. e - l Repre...
Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-fold upregulation of PAD4 protein expression in these areas (Fig. ). To test the hypothesis that increased release of NETs, orchestrated by PAD4, participates in stroke recovery, we first studied the role of overexpression of PAD4 on vascular remodeling. Immunohistochemical analysis indicated extensive expression of recombinant adeno-PAD4-flag-infected cells in the cortex at 4 days after injection (Fig. ). Overexpression of PAD4 in the cortex was also confirmed by immunoblotting (Supplementary Fig. ). PAD4-flag was present in the neurons, neutrophils, macrophages/microglial cells, and microglial cells, but was rarely detected in astrocytes (Supplementary Fig. ). Injection of PAD4 adenovirus in ischemic mice produced ~2.6-fold more NETs than control virus (Fig. and Supplementary Fig. ). We then observed a significant increase in the n...
To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig. ). Thus, this approach allowed us to provide evidence that effects of PAD4 deficiency on long-term outcomes are not secondary to the reduced lesion size. PAD4 deficiency did not alter the number of infiltrating neutrophils in the ischemic brain tissue (Supplementary Fig. ), whereas the levels of H3Cit in the lysates from the ischemic cortex at 3 days were greatly reduced in PAD4 -/- mice and mice receiving Cl-amidine (Fig. and Supplementary Fig. ). Similarly, examination of the brain tissues revealed that PAD4 deficiency or Cl-amidine reduced Sytox green-positive extracellular DNA fibers (Fig. ) in the ischemic cortex. Previous studies have shown that PAD4 may also function in other cells,. We found that PAD4 deficiency or Cl-amidine substantially reduced neutrophil NETs, as seen by the decrease in H3Cit + neutrophils (Fig....
The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly increased levels of STING (Fig. and Supplementary Fig. ) and robust induction of phosphorylated TANK-binding kinase 1 (pTBK1) and TBK1-dependent IFN regulatory factor 3 (IRF3) activation (Fig. and Supplementary Fig. ). Treatment with the PAD inhibitor Cl-amidine reduced the levels of stroke-induced STING-meditated signaling in the cortical areas compared with vehicle-treated mice (Fig. and Supplementary Fig. ). Neutrophil depletion also resulted in a significant reduction in the levels of STING and the STING downstream signaling molecules including pTBK1, pIRF3, and IFN-β in the ischemic brain relative to IgG-treated controls (Fig. and Supplementary Fig. ). To test whether the signaling part mediated by STING is related to neutrophils, we isolated neutrophils from bone marrow of ischemic mice and stimulated them with...
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used as controls. All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light-dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%. Focal cortical cerebral ischemia was induced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA),. Male mice weighing 23-26 g were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. After making a 2 cm curved skin incision between the right eye and the right ear using surgical scissors, the temporal muscle was retracted laterally. Under an operating microscope, a 1.5 mm-diameter window was opened using a high-speed micro drill (Stoelting, CellPoint Scientific, Maryland)...
Machine-readable layer
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"name": "Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke methods",
"description": "Evidence-backed execution summary for Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke methods from Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke.",
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"name": "Stroke induces NET formation",
"text": "To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (Fig. ). In line with this, elevated levels of circulating DNA was found in the plasma from these mice (Fig. ). We then isolated neutrophils from the peripheral blood of these mice and incubated them with or without lipopolysaccharide (LPS) stimulation. We found that either unstimulated or LPS-stimulated neutrophils from ischemic mice showed a significant increase in H3Cit + neutrophils and NET formation (Fig. ), indicating that neutrophils from mice subjected to stroke are primed to undergo NETosis. Fig. 3 Neutrophils form NETs presenting in the brain after..."
},
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"name": "Stroke induces NET formation",
"text": "As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed an increased amount of H3Cit that was most robust from 3-5 days (Fig. ), suggesting that NETs may play an important role during the delayed phases after stroke. Immunostaining revealed that the peri-infarct cortex was extensively labeled with H3Cit + cells at 3 days (Fig. ). To identify which type of cells expressed H3cit after stroke, double immunofluorescence with confocal microscopy was performed on brain sections. This analysis revealed that H3Cit was colocalized with Ly6G-positive neutrophils, F4/80-positive macrophages/microglial cells, Iba1-positive microglial cells, NeuN-positive neurons, and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. ). Importantly, 78.7% of..."
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"position": 3,
"name": "Disruption of NETs enhances vascular remodeling after stroke",
"text": "We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrophils in the peri-infarct cortical areas (Supplementary Fig. ) but reduced H3Cit levels (Fig. ). Compared with the vehicle controls, administration of DNase 1 significantly reduced BBB permeability (Fig. ) and extravascular IgG deposits (Fig. ), and enhanced both Pdgfr-β + and CD13 + pericyte coverage on brain microvessels (Fig. and Supplementary Fig. ). Vascular branches (Supplementary Fig. ), microvascular length (Fig. ), perfused capillary length (Fig. ), and tomato-lectin perfused vessels (Fig. ) were also increased in the brains of mice treated with DNase 1. Next, we investigated the importance of neutrophil NETs in vascular remodeling. Our re..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Disruption of NETs enhances vascular remodeling after stroke",
"text": "Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and perfused cortical vessels (Fig. ) in anti-Ly6G antibody-treated mice compared with control IgG-treated mice. Treatment with DNase 1 starting at 7 days after stroke also attenuated BBB disruption (Fig. ), increased microvessels (Fig. ), and improved capillary perfusion (Fig. ) at 14 days. Fig. 5 Increased vascular remodeling by delayed inhibition of NET formation. a - d Representative confocal images ( a, c ) and quantitative analysis of IgG extravascular deposits ( b, d ) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "PAD4 regulates BBB permeability and neovascularization",
"text": "Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-fold upregulation of PAD4 protein expression in these areas (Fig. ). To test the hypothesis that increased release of NETs, orchestrated by PAD4, participates in stroke recovery, we first studied the role of overexpression of PAD4 on vascular remodeling. Immunohistochemical analysis indicated extensive expression of recombinant adeno-PAD4-flag-infected cells in the cortex at 4 days after injection (Fig. ). Overexpression of PAD4 in the cortex was also confirmed by immunoblotting (Supplementary Fig. ). PAD4-flag was present in the neurons, neutrophils, macro..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "PAD4 regulates BBB permeability and neovascularization",
"text": "To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig. ). Thus, this approach allowed us to provide evidence that effects of PAD4 deficiency on long-term outcomes are not secondary to the reduced lesion size. PAD4 deficiency did not alter the number of infiltrating neutrophils in the ischemic brain tissue (Supplementary Fig. ), whereas the levels of H3Cit in the lysates from the ischemic cortex at 3 days were greatly reduced in PAD4 -/- mice and mice receiving Cl-amidine (Fig. and Supplementary Fig. ). Similarly, examination of the brain tissues revealed that PAD4 deficiency or Cl-amidine reduced..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "NETs are responsible for STING-mediated vascular remodeling",
"text": "The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly increased levels of STING (Fig. and Supplementary Fig. ) and robust induction of phosphorylated TANK-binding kinase 1 (pTBK1) and TBK1-dependent IFN regulatory factor 3 (IRF3) activation (Fig. and Supplementary Fig. ). Treatment with the PAD inhibitor Cl-amidine reduced the levels of stroke-induced STING-meditated signaling in the cortical areas compared with vehicle-treated mice (Fig. and Supplementary Fig. ). Neutrophil depletion also resulted in a significant reduction in the levels of STING and the STING downstream signaling molecules..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Methods",
"text": "Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used as controls. All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light-dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%. Focal cortical cerebral ischemia was induced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA),. Male mice weighing 23-26 g were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Afte..."
}
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"name": "In-vivo multiphoton microscopy"
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"name": "Neutrophil depletion"
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"name": "Blood counts"
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"name": "In-vivo multiphoton microscopy"
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"name": "Stroke induces NET formation"
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"name": "Disruption of NETs enhances vascular remodeling after stroke"
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"name": "Disruption of NETs enhances vascular remodeling after stroke"
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"name": "NETs are responsible for STING-mediated vascular remodeling"
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"headline": "Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke",
"datePublished": "2020",
"author": [
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"name": "Yuanbo Zhu"
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"name": "Xiaofei Bai"
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{
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"name": "Ranran Wang"
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"name": "Yongliang Cao"
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"name": "Haochen Xu"
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"name": "Haiyu Luo"
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"name": "Lu Lu"
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"name": "Mei-Juan Shi"
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"name": "Yujing Tian"
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"name": "Wenying Fan"
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"name": "Bing-Qiao Zhao"
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"identifier": "10.1038/s41467-020-16191-y"
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