Interleukin-4 is Essential for Microglia/Macrophage M2 Polarization and Long-term Recovery after Cerebral Ischemia methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Materials and Methods
All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Wild-type (WT) or IL-4 knockout (KO) male C57/BL6 mice (8-10w, 25-30 g body weight; Jackson Laboratory, Bar Harbor, Maine, USA) were randomly assigned to sham or cerebral ischemia groups using a lottery drawing box. Transient focal ischemia was induced by MCAO for 60 minutes as previously described. Sham-operated animals underwent anesthesia and exposure of the arteries without MCAO induction. Rectal temperature was maintained at 37.0°C±0.5°C during and after surgery with a temperature-controlled heating pad. Physiological parameters were maintained within normal ranges. Regional cerebral blood flow (rCBF...
Intracerebroventricular IL-4 administration
WT mice subjected to 60 min MCAO or sham operation were randomly assigned to vehicle or IL-4 groups. An intracerebroventricular (icv) catheter was stereotaxically implanted into the ventricle contralateral to the lesion site (coordinates: -0.20 mm anterior and 1.00 mm lateral to bregma). An ALZET osmotic minipump containing IL-4 (Peprotech, 60 ng/d) or phosphate-buffered saline (PBS) was implanted subcutaneously above the spine and connected to the icv catheter by an investigator blinded to experimental groups. The pump was programmed to achieve a constant infusion rate of 0.5 µl/hr, starting from 6h after tMCAO. The pump was removed at 7d after tMCAO.
Loss of IL-4 disrupts long-term neurological functions after ischemic/reperfusion injury
Next we used IL-4 KO mice to investigate the effect of IL-4 on long-term neurological outcomes after stroke. Transient focal ischemia was induced by 60 min tMCAO. Physiological parameters, including blood pressure, blood gases, and glucose levels, exhibited no significant differences between WT and KO mice (not shown). rCBF, as determined with a laser speckle contrast imager, did not differ by genotype before ischemia, 5 min after ischemia, or 5 min after reperfusion ( ). These findings verify that WT and IL-4 KO mice were subjected to the same ischemic insult during tMCAO and that any differences across genotypes cannot be attributed to variations in blood flow.
Loss of IL-4 disrupts long-term neurological functions after ischemic/reperfusion injury
We further determined whether IL-4 deficiency impaired long-term cognitive functions using the Morris water maze test. The IL-4 KO and WT mice exhibited comparable cognitive functions after sham surgery under physiological conditions. However, long-term learning and memory deficits were significantly exacerbated in IL-4 KO mice after tMCAO, as manifested by an increased latency to find the hidden platform (impaired spatial learning ability) and reduced time spent in the target quadrant when the platform was removed (impaired memory) compared to WT mice ( ). Both genotypes exhibited similar swim speeds ( ), suggesting equivalent swimming skills. Taken together, the results of the behavioral assays demonstrate a stable or persistent exacerbation of neurological dysfunction after stroke in the absence of IL-4.
IL-4 deficiency exacerbates long term neurological functions and impairs microglia/macrophage M2 polarization after p...
Effects that can be generalized across different stroke models are more likely to translate to clinical studies. Thus, we also tested the effect of IL-4 in a model of permanent distal MCAO (dMCAO). The corner test revealed that dMCAO resulted in transient sensorimotor deficits early after surgery with a recovery by 9d after dMCAO in WT mice. IL-4 deficiency significantly prolonged the sensorimotor deficits ( ). Consistent with the results in the tMCAO model, NeuN immunostaining revealed similar neuronal tissue loss in WT and IL-4 KO mice at 21d after dMCAO ( ). RT-PCR measurements revealed increased mRNA expression of M1 markers (TNFα) and reduced expression of M2 markers (CD206 and IL-10) at 21d after dMCAO in IL-4 KO mice ( ). These results verify that IL-4 deficiency impairs M2 polarization and worsens neurological outcomes in two independent stroke models.
Administration of IL-4 improves neurological functions after stroke without reducing neuronal loss
If loss of endogenous IL-4 leads to long-term neurological dysfunction, administration of exogenous IL-4 should have the converse effect and lead to stable recovery of function. Furthermore, if IL-4 protein levels are low 14d after stroke injury but receptor levels are high, then one would expect IL-4 supplementation to be extremely effective at this time point. Thus, to further confirm the importance of IL-4 in persistent stroke recovery, recombinant IL-4 (60 ng/d) was continuously infused through an osmotic minipump into the brain of C57/BL6 mice beginning 6h after tMCAO or sham operation and lasting for one week. Neurological functions were then assessed at various time points after surgery. IL-4 post-treatment failed to improve neurobehavioral performance 1-7 d after tMCAO. However, at late stages after tMCAO, IL-4-treated mice showed improved performance in the Rotarod (14&...
Measurement outputs
What raw and processed outputs should exist?
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (6)
All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Wild-type (WT) or IL-4 knockout (KO) male C57/BL6 mice (8-10w, 25-30 g body weight; Jackson Laboratory, Bar Harbor, Maine, USA) were randomly assigned to sham or cerebral ischemia groups using a lottery drawing box. Transient focal ischemia was induced by MCAO for 60 minutes as previously described. Sham-operated animals underwent anesthesia and exposure of the arteries without MCAO induction. Rectal temperature was maintained at 37.0°C±0.5°C during and after surgery with a temperature-controlled heating pad. Physiological parameters were maintained within normal ranges. Regional cerebral blood flow (rCBF) was monitored in all stroke animals using laser Doppler flowmetry. Animals that died or failed to show at least 70% rCBF reduction were excluded from further analyses. Mice in both genotypes were subjected to repeated measurements of CBF before ischemia, 5 min after tMCAO, and 5 min after reperfus...
WT mice subjected to 60 min MCAO or sham operation were randomly assigned to vehicle or IL-4 groups. An intracerebroventricular (icv) catheter was stereotaxically implanted into the ventricle contralateral to the lesion site (coordinates: -0.20 mm anterior and 1.00 mm lateral to bregma). An ALZET osmotic minipump containing IL-4 (Peprotech, 60 ng/d) or phosphate-buffered saline (PBS) was implanted subcutaneously above the spine and connected to the icv catheter by an investigator blinded to experimental groups. The pump was programmed to achieve a constant infusion rate of 0.5 µl/hr, starting from 6h after tMCAO. The pump was removed at 7d after tMCAO.
Next we used IL-4 KO mice to investigate the effect of IL-4 on long-term neurological outcomes after stroke. Transient focal ischemia was induced by 60 min tMCAO. Physiological parameters, including blood pressure, blood gases, and glucose levels, exhibited no significant differences between WT and KO mice (not shown). rCBF, as determined with a laser speckle contrast imager, did not differ by genotype before ischemia, 5 min after ischemia, or 5 min after reperfusion ( ). These findings verify that WT and IL-4 KO mice were subjected to the same ischemic insult during tMCAO and that any differences across genotypes cannot be attributed to variations in blood flow.
We further determined whether IL-4 deficiency impaired long-term cognitive functions using the Morris water maze test. The IL-4 KO and WT mice exhibited comparable cognitive functions after sham surgery under physiological conditions. However, long-term learning and memory deficits were significantly exacerbated in IL-4 KO mice after tMCAO, as manifested by an increased latency to find the hidden platform (impaired spatial learning ability) and reduced time spent in the target quadrant when the platform was removed (impaired memory) compared to WT mice ( ). Both genotypes exhibited similar swim speeds ( ), suggesting equivalent swimming skills. Taken together, the results of the behavioral assays demonstrate a stable or persistent exacerbation of neurological dysfunction after stroke in the absence of IL-4.
Effects that can be generalized across different stroke models are more likely to translate to clinical studies. Thus, we also tested the effect of IL-4 in a model of permanent distal MCAO (dMCAO). The corner test revealed that dMCAO resulted in transient sensorimotor deficits early after surgery with a recovery by 9d after dMCAO in WT mice. IL-4 deficiency significantly prolonged the sensorimotor deficits ( ). Consistent with the results in the tMCAO model, NeuN immunostaining revealed similar neuronal tissue loss in WT and IL-4 KO mice at 21d after dMCAO ( ). RT-PCR measurements revealed increased mRNA expression of M1 markers (TNFα) and reduced expression of M2 markers (CD206 and IL-10) at 21d after dMCAO in IL-4 KO mice ( ). These results verify that IL-4 deficiency impairs M2 polarization and worsens neurological outcomes in two independent stroke models.
If loss of endogenous IL-4 leads to long-term neurological dysfunction, administration of exogenous IL-4 should have the converse effect and lead to stable recovery of function. Furthermore, if IL-4 protein levels are low 14d after stroke injury but receptor levels are high, then one would expect IL-4 supplementation to be extremely effective at this time point. Thus, to further confirm the importance of IL-4 in persistent stroke recovery, recombinant IL-4 (60 ng/d) was continuously infused through an osmotic minipump into the brain of C57/BL6 mice beginning 6h after tMCAO or sham operation and lasting for one week. Neurological functions were then assessed at various time points after surgery. IL-4 post-treatment failed to improve neurobehavioral performance 1-7 d after tMCAO. However, at late stages after tMCAO, IL-4-treated mice showed improved performance in the Rotarod (14-35d) and foot fault tests (21-35d) ( ). Moreover, IL-4-treated mice exhibited improvements in cognitive functions, as manifested by improved spatial learning and memory in the Morris water maze ( ). Overall neuronal tissue loss was measured on NeuN-stained sections at 14d and 35d after t...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Interleukin-4 is Essential for Microglia/Macrophage M2 Polarization and Long-term Recovery after Cerebral Ischemia methods",
"description": "Protocol for Interleukin-4 is Essential for Microglia/Macrophage M2 Polarization and Long-term Recovery after Cerebral Ischemia methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Materials and Methods",
"text": "All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Wild-type (WT) or IL-4 knockout (KO) male C57/BL6 mice (8-10w, 25-30 g body weight; Jackson Laboratory, Bar Harbor, Maine, USA) were randomly assigned to sham or cerebral ischemia groups using a lottery drawing box. Transient focal ischemia was induced by MCAO for 60 minutes as previously described. Sham-operated animals underwent anesthesia and exposure of the arteries without MCAO induction. Rectal temperature was maintained at 37.0°C±0.5°C during and after surgery with a temperature-controlled heating pad. Physiological parameters were maintained within normal ranges. Regional cerebral blood flow (rCBF..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Intracerebroventricular IL-4 administration",
"text": "WT mice subjected to 60 min MCAO or sham operation were randomly assigned to vehicle or IL-4 groups. An intracerebroventricular (icv) catheter was stereotaxically implanted into the ventricle contralateral to the lesion site (coordinates: -0.20 mm anterior and 1.00 mm lateral to bregma). An ALZET osmotic minipump containing IL-4 (Peprotech, 60 ng/d) or phosphate-buffered saline (PBS) was implanted subcutaneously above the spine and connected to the icv catheter by an investigator blinded to experimental groups. The pump was programmed to achieve a constant infusion rate of 0.5 µl/hr, starting from 6h after tMCAO. The pump was removed at 7d after tMCAO."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Loss of IL-4 disrupts long-term neurological functions after ischemic/reperfusion injury",
"text": "Next we used IL-4 KO mice to investigate the effect of IL-4 on long-term neurological outcomes after stroke. Transient focal ischemia was induced by 60 min tMCAO. Physiological parameters, including blood pressure, blood gases, and glucose levels, exhibited no significant differences between WT and KO mice (not shown). rCBF, as determined with a laser speckle contrast imager, did not differ by genotype before ischemia, 5 min after ischemia, or 5 min after reperfusion ( ). These findings verify that WT and IL-4 KO mice were subjected to the same ischemic insult during tMCAO and that any differences across genotypes cannot be attributed to variations in blood flow."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Loss of IL-4 disrupts long-term neurological functions after ischemic/reperfusion injury",
"text": "We further determined whether IL-4 deficiency impaired long-term cognitive functions using the Morris water maze test. The IL-4 KO and WT mice exhibited comparable cognitive functions after sham surgery under physiological conditions. However, long-term learning and memory deficits were significantly exacerbated in IL-4 KO mice after tMCAO, as manifested by an increased latency to find the hidden platform (impaired spatial learning ability) and reduced time spent in the target quadrant when the platform was removed (impaired memory) compared to WT mice ( ). Both genotypes exhibited similar swim speeds ( ), suggesting equivalent swimming skills. Taken together, the results of the behavioral assays demonstrate a stable or persistent exacerbation of neurological dysfunction after stroke in the absence of IL-4."
},
{
"@type": "HowToStep",
"position": 5,
"name": "IL-4 deficiency exacerbates long term neurological functions and impairs microglia/macrophage M2 polarization after p...",
"text": "Effects that can be generalized across different stroke models are more likely to translate to clinical studies. Thus, we also tested the effect of IL-4 in a model of permanent distal MCAO (dMCAO). The corner test revealed that dMCAO resulted in transient sensorimotor deficits early after surgery with a recovery by 9d after dMCAO in WT mice. IL-4 deficiency significantly prolonged the sensorimotor deficits ( ). Consistent with the results in the tMCAO model, NeuN immunostaining revealed similar neuronal tissue loss in WT and IL-4 KO mice at 21d after dMCAO ( ). RT-PCR measurements revealed increased mRNA expression of M1 markers (TNFα) and reduced expression of M2 markers (CD206 and IL-10) at 21d after dMCAO in IL-4 KO mice ( ). These results verify that IL-4 deficiency impairs M2 polarization and worsens neurological outcomes in two independent stroke models."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Administration of IL-4 improves neurological functions after stroke without reducing neuronal loss",
"text": "If loss of endogenous IL-4 leads to long-term neurological dysfunction, administration of exogenous IL-4 should have the converse effect and lead to stable recovery of function. Furthermore, if IL-4 protein levels are low 14d after stroke injury but receptor levels are high, then one would expect IL-4 supplementation to be extremely effective at this time point. Thus, to further confirm the importance of IL-4 in persistent stroke recovery, recombinant IL-4 (60 ng/d) was continuously infused through an osmotic minipump into the brain of C57/BL6 mice beginning 6h after tMCAO or sham operation and lasting for one week. Neurological functions were then assessed at various time points after surgery. IL-4 post-treatment failed to improve neurobehavioral performance 1-7 d after tMCAO. However, at late stages after tMCAO, IL-4-treated mice showed improved performance in the Rotarod (14&..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Interleukin-4 is Essential for Microglia/Macrophage M2 Polarization and Long-term Recovery after Cerebral Ischemia",
"datePublished": "2016",
"author": [
{
"@type": "Person",
"name": "Xiangrong Liu"
},
{
"@type": "Person",
"name": "Jia Liu"
},
{
"@type": "Person",
"name": "Shangfeng Zhao"
},
{
"@type": "Person",
"name": "Haiyue Zhang"
},
{
"@type": "Person",
"name": "Wei Cai"
},
{
"@type": "Person",
"name": "Mengfei Cai"
},
{
"@type": "Person",
"name": "Xunming Ji"
},
{
"@type": "Person",
"name": "Rehana K. Leak"
},
{
"@type": "Person",
"name": "Yanqin Gao"
},
{
"@type": "Person",
"name": "Jun Chen"
},
{
"@type": "Person",
"name": "Xiaoming Hu"
}
],
"identifier": "10.1038/nn.3469"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Interleukin-4 is Essential for Microglia/Macrophage M2 Polarization and Long-term Recovery after Cerebral Ischemia methods",
"item": "https://replicatescience.com/experiments/interleukin-4-is-essential-for-microglia-macrophage-m2-polarization-and-long-term-recovery-after-cerebral-ischemia-methods-xiangrong-liu-pmc4729613/interleukin-4-is-essential-for-microglia-macrophage-m2-polarization-and-long-term-recovery-after-cer-mlpgx4p5"
}
]
}
]