Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice 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?
Animal treatments
All rodent experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at the University of California, Irvine (UCI). LPS treatment: LPS (from Escherichia coli 055:B5; Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 0.1 mg/ml and administered intraperitoneally (IP) at a dose of 0.5 mg/kg body weight. Following any treatments, mice were sacrificed and brains isolated. One-half of the brain was fixed in 4 % paraformaldehyde and the other half was snap frozen on dry ice and stored at -80 °C until analysis.
Behavioral testing
Open field: Open-field testing was employed as a measure of anxiety as well as motor ability. Mice were placed in an opaque white box (33.7 cm L × 27.3 cm W × 21.6 cm H) for 5 min while their behavior was video-recorded. The amount of time spent in the center versus the perimeter of the box and motor readouts (distance moved and velocity) were obtained. Barnes maze: Mice were tested in the Barnes maze (table diameter 122 cm, 40 holes with diameter 4.8 cm, elevated 140 cm above the ground) for 5 days (acquisition, 4 days, 2 trials/day, maximum 120 s/trial, 15 min intertrial interval; 24-hr probe, 1 day, 1 trial, maximum of 120 s), as a measure of spatial learning and memory. Latency to find the target and enter the target box (i.e., escape latency) was recorded live each day of acquisit...
Statistics
Appropriate statistical analyses were carried out to determine significance between groups using unpaired Student's t test for comparisons between two groups, one-way ANOVA for multiple comparisons, with Newman-Keuls post hoc multiple comparison test. Multiple-day behavioral data and MSD® Multi-Spot Assay data were analyzed using a two-way ANOVA (treatment x day of testing and diet x injection, respectively) using the MIXED procedure of the Statistical Analysis Systems software (SAS Institute Inc.). For behavioral data, "mouse" was a random effect and "day of testing" was a repeated measure. Post hoc paired contrasts were used to examine biologically relevant interactions from the two-way ANOVA.
Microglial depletion does not affect behavior or learning and memory
Two-month-old wild-type mice were treated with either vehicle or 300- or 1200 mg/kg chow PLX5622 for 14 days, and behavioral analyses were then conducted ( n = 10 per group) (Fig. ). Mice were first tested in open field analyses. No differences were seen in mice with either dose of PLX5622 in the distance traveled (Fig. ), velocity (Fig. ), time spent in the open area (Fig. ), or time spent in the edge of the arena (Fig. ), compared to vehicle-treated mice. Next, mice were tested on Barnes maze-a hippocampal-dependent learning and memory task. Again, no differences were found between any of the groups on acquisition of the time to find the escape hole (Fig. ) or in the probe trial conducted 24 h after the last training session (Fig. ). Thus, depletion of microglia with PLX5622 does not induce any deficits in t...
Lower-dose PLX5622 treatment partially reduces microglial numbers
We next examined the brains of the 3xTg-AD mice treated for 3 months to evaluate the effects of PLX5622 on glial cells and pathology. Immunohistochemistry revealed 30 % reductions in microglial numbers in areas not adjacent to plaque loads (Fig. ), in line with the data in Fig., showing that we can achieve sustained targeted reductions in brainwide microglia, rather than just total elimination. Microglia in the PLX5622-treated mice had significantly larger cell sizes (Fig. ), in line with our previous findings [ ], and also reduced IBA1 staining intensity (Fig. ). Immunostaining for the astrocytic markers GFAP and S100 showed robust astrogliosis associated with plaques (Fig. ), but PLX5622 treatment did not alter this response or the overall number of GFAP/S100 + cells present within the hippocampus (Fig. ). Inflammatory profiling using m...
Lower-dose PLX5622 treatment prevents microglial association with plaques
Sandwich ELISA for Aβ40 and Aβ42 in the soluble and insoluble fractions revealed no significant differences between the PLX5622-treated group and the controls (Fig. ). Thio-S staining revealed abundant dense core plaques in all animals, particularly in the subiculum (Fig. ). Quantification of number of plaques (Fig. ), average plaque size (Fig. ), or distribution of small, medium, or large plaques (Fig. ), revealed no changes with treatment. Fig. 6 Lower-dose CSF1R inhibition does not alter Aβ or Tau levels. a, b Sandwich ELISA in soluble and insoluble fraction revealed no significant differences in Aβ40 or Aβ42 between groups. c, d Thioflavin-S staining was performed, revealing plaques in both control and treated groups. Of the subiculum, 10 × images are shown. e - g Analysis of plaques revealed no significant differe...
Lower-dose PLX5622 treatment prevents microglial association with plaques
However, stark differences were seen in the microglia that are associated with plaques-in vehicle-treated animals, microglia were densely packed around plaques and displayed an activated morphology (Fig. ). Mice treated with PLX5622 showed comparable plaque load, but microglia were not associated with plaques to the same extent (Fig. ), suggesting that low doses of PLX5622 do not fully eliminate microglia but alter their response to inflammatory stimuli, such as plaques. Indeed, closeup z-stacks showed a clear association of microglia with plaques in untreated 3xTg-AD mice (Fig. ) but a lack of association in PLX5622-treated mice (Fig. ). Quantification of the number of microglia associated with a plaque and normalized to plaque diameter revealed a 70 % reduction in microglia associated with plaques. To explore the possibility that this lower dose of...
Lower-dose PLX5622 treatment prevents microglial association with plaques
3xTg-AD mice also show progressive tau pathology as they age, including somatodendritic accumulation of human tau and hyperphosphorylation [ ]. Quantification of total human tau accumulation within somatodendritic compartments of CA1 neurons showed no differences with treatment, nor tau phosphorylated at S202/T205 (Fig. ).
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 (8)
All rodent experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at the University of California, Irvine (UCI). LPS treatment: LPS (from Escherichia coli 055:B5; Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 0.1 mg/ml and administered intraperitoneally (IP) at a dose of 0.5 mg/kg body weight. Following any treatments, mice were sacrificed and brains isolated. One-half of the brain was fixed in 4 % paraformaldehyde and the other half was snap frozen on dry ice and stored at -80 °C until analysis.
Open field: Open-field testing was employed as a measure of anxiety as well as motor ability. Mice were placed in an opaque white box (33.7 cm L × 27.3 cm W × 21.6 cm H) for 5 min while their behavior was video-recorded. The amount of time spent in the center versus the perimeter of the box and motor readouts (distance moved and velocity) were obtained. Barnes maze: Mice were tested in the Barnes maze (table diameter 122 cm, 40 holes with diameter 4.8 cm, elevated 140 cm above the ground) for 5 days (acquisition, 4 days, 2 trials/day, maximum 120 s/trial, 15 min intertrial interval; 24-hr probe, 1 day, 1 trial, maximum of 120 s), as a measure of spatial learning and memory. Latency to find the target and enter the target box (i.e., escape latency) was recorded live each day of acquisition, while latency to find the target and number of errors prior to finding the target was recorded live on the probe day. Morris water maze: Hidden platform Morris water maze (MWM) training and testing were conducted as described previously [ ]. Novel object and place recognition tasks: Novel obje...
Appropriate statistical analyses were carried out to determine significance between groups using unpaired Student's t test for comparisons between two groups, one-way ANOVA for multiple comparisons, with Newman-Keuls post hoc multiple comparison test. Multiple-day behavioral data and MSD® Multi-Spot Assay data were analyzed using a two-way ANOVA (treatment x day of testing and diet x injection, respectively) using the MIXED procedure of the Statistical Analysis Systems software (SAS Institute Inc.). For behavioral data, "mouse" was a random effect and "day of testing" was a repeated measure. Post hoc paired contrasts were used to examine biologically relevant interactions from the two-way ANOVA.
Two-month-old wild-type mice were treated with either vehicle or 300- or 1200 mg/kg chow PLX5622 for 14 days, and behavioral analyses were then conducted ( n = 10 per group) (Fig. ). Mice were first tested in open field analyses. No differences were seen in mice with either dose of PLX5622 in the distance traveled (Fig. ), velocity (Fig. ), time spent in the open area (Fig. ), or time spent in the edge of the arena (Fig. ), compared to vehicle-treated mice. Next, mice were tested on Barnes maze-a hippocampal-dependent learning and memory task. Again, no differences were found between any of the groups on acquisition of the time to find the escape hole (Fig. ) or in the probe trial conducted 24 h after the last training session (Fig. ). Thus, depletion of microglia with PLX5622 does not induce any deficits in these particular tasks, in agreement with our previous data [ ]. Fig. 2 CSF1R inhibition does not alter cognition or behavior in adult mice. a Two-month-old wild-type mice were treated with either vehicle, 300 or 1200 mg/kg PLX5622 for 14 days, and behavioral analyses were conducted using a...
We next examined the brains of the 3xTg-AD mice treated for 3 months to evaluate the effects of PLX5622 on glial cells and pathology. Immunohistochemistry revealed 30 % reductions in microglial numbers in areas not adjacent to plaque loads (Fig. ), in line with the data in Fig., showing that we can achieve sustained targeted reductions in brainwide microglia, rather than just total elimination. Microglia in the PLX5622-treated mice had significantly larger cell sizes (Fig. ), in line with our previous findings [ ], and also reduced IBA1 staining intensity (Fig. ). Immunostaining for the astrocytic markers GFAP and S100 showed robust astrogliosis associated with plaques (Fig. ), but PLX5622 treatment did not alter this response or the overall number of GFAP/S100 + cells present within the hippocampus (Fig. ). Inflammatory profiling using multiplex technology in soluble brain homogenates showed no changes in Il-1β or Il-6 with treatment, in line with the data obtained following LPS injections (Fig. ), but significant increases were found in CXCL1 and TNFα (Fig. ). Markers IFN-γ, Il-10, Il-12p70, Il-2, Il-4,...
Sandwich ELISA for Aβ40 and Aβ42 in the soluble and insoluble fractions revealed no significant differences between the PLX5622-treated group and the controls (Fig. ). Thio-S staining revealed abundant dense core plaques in all animals, particularly in the subiculum (Fig. ). Quantification of number of plaques (Fig. ), average plaque size (Fig. ), or distribution of small, medium, or large plaques (Fig. ), revealed no changes with treatment. Fig. 6 Lower-dose CSF1R inhibition does not alter Aβ or Tau levels. a, b Sandwich ELISA in soluble and insoluble fraction revealed no significant differences in Aβ40 or Aβ42 between groups. c, d Thioflavin-S staining was performed, revealing plaques in both control and treated groups. Of the subiculum, 10 × images are shown. e - g Analysis of plaques revealed no significant difference between treated and untreated brains in average number of plaques, average plaque size, or distribution of large, medium, and small plaques. h - k Immunostaining for total human tau with HT7 ( h, i ) and AT8 tau ( j, k ) reveals no significant differences in tau levels with PLX5622 treat...
However, stark differences were seen in the microglia that are associated with plaques-in vehicle-treated animals, microglia were densely packed around plaques and displayed an activated morphology (Fig. ). Mice treated with PLX5622 showed comparable plaque load, but microglia were not associated with plaques to the same extent (Fig. ), suggesting that low doses of PLX5622 do not fully eliminate microglia but alter their response to inflammatory stimuli, such as plaques. Indeed, closeup z-stacks showed a clear association of microglia with plaques in untreated 3xTg-AD mice (Fig. ) but a lack of association in PLX5622-treated mice (Fig. ). Quantification of the number of microglia associated with a plaque and normalized to plaque diameter revealed a 70 % reduction in microglia associated with plaques. To explore the possibility that this lower dose of CSF1R inhibitor could be selectively killing plaque-associated microglia, we performed immunostaining for active caspase-3, as an indicator of cell death. However, no increased microglial cell death was seen in PLX5622-treated mice in areas adjacent to plaques (data not shown). This suggests that t...
3xTg-AD mice also show progressive tau pathology as they age, including somatodendritic accumulation of human tau and hyperphosphorylation [ ]. Quantification of total human tau accumulation within somatodendritic compartments of CA1 neurons showed no differences with treatment, nor tau phosphorylated at S202/T205 (Fig. ).
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice methods",
"description": "Protocol for Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Animal treatments",
"text": "All rodent experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee at the University of California, Irvine (UCI). LPS treatment: LPS (from Escherichia coli 055:B5; Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 0.1 mg/ml and administered intraperitoneally (IP) at a dose of 0.5 mg/kg body weight. Following any treatments, mice were sacrificed and brains isolated. One-half of the brain was fixed in 4 % paraformaldehyde and the other half was snap frozen on dry ice and stored at -80 °C until analysis."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Behavioral testing",
"text": "Open field: Open-field testing was employed as a measure of anxiety as well as motor ability. Mice were placed in an opaque white box (33.7 cm L × 27.3 cm W × 21.6 cm H) for 5 min while their behavior was video-recorded. The amount of time spent in the center versus the perimeter of the box and motor readouts (distance moved and velocity) were obtained. Barnes maze: Mice were tested in the Barnes maze (table diameter 122 cm, 40 holes with diameter 4.8 cm, elevated 140 cm above the ground) for 5 days (acquisition, 4 days, 2 trials/day, maximum 120 s/trial, 15 min intertrial interval; 24-hr probe, 1 day, 1 trial, maximum of 120 s), as a measure of spatial learning and memory. Latency to find the target and enter the target box (i.e., escape latency) was recorded live each day of acquisit..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Statistics",
"text": "Appropriate statistical analyses were carried out to determine significance between groups using unpaired Student's t test for comparisons between two groups, one-way ANOVA for multiple comparisons, with Newman-Keuls post hoc multiple comparison test. Multiple-day behavioral data and MSD® Multi-Spot Assay data were analyzed using a two-way ANOVA (treatment x day of testing and diet x injection, respectively) using the MIXED procedure of the Statistical Analysis Systems software (SAS Institute Inc.). For behavioral data, \"mouse\" was a random effect and \"day of testing\" was a repeated measure. Post hoc paired contrasts were used to examine biologically relevant interactions from the two-way ANOVA."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Microglial depletion does not affect behavior or learning and memory",
"text": "Two-month-old wild-type mice were treated with either vehicle or 300- or 1200 mg/kg chow PLX5622 for 14 days, and behavioral analyses were then conducted ( n = 10 per group) (Fig. ). Mice were first tested in open field analyses. No differences were seen in mice with either dose of PLX5622 in the distance traveled (Fig. ), velocity (Fig. ), time spent in the open area (Fig. ), or time spent in the edge of the arena (Fig. ), compared to vehicle-treated mice. Next, mice were tested on Barnes maze-a hippocampal-dependent learning and memory task. Again, no differences were found between any of the groups on acquisition of the time to find the escape hole (Fig. ) or in the probe trial conducted 24 h after the last training session (Fig. ). Thus, depletion of microglia with PLX5622 does not induce any deficits in t..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Lower-dose PLX5622 treatment partially reduces microglial numbers",
"text": "We next examined the brains of the 3xTg-AD mice treated for 3 months to evaluate the effects of PLX5622 on glial cells and pathology. Immunohistochemistry revealed 30 % reductions in microglial numbers in areas not adjacent to plaque loads (Fig. ), in line with the data in Fig., showing that we can achieve sustained targeted reductions in brainwide microglia, rather than just total elimination. Microglia in the PLX5622-treated mice had significantly larger cell sizes (Fig. ), in line with our previous findings [ ], and also reduced IBA1 staining intensity (Fig. ). Immunostaining for the astrocytic markers GFAP and S100 showed robust astrogliosis associated with plaques (Fig. ), but PLX5622 treatment did not alter this response or the overall number of GFAP/S100 + cells present within the hippocampus (Fig. ). Inflammatory profiling using m..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Lower-dose PLX5622 treatment prevents microglial association with plaques",
"text": "Sandwich ELISA for Aβ40 and Aβ42 in the soluble and insoluble fractions revealed no significant differences between the PLX5622-treated group and the controls (Fig. ). Thio-S staining revealed abundant dense core plaques in all animals, particularly in the subiculum (Fig. ). Quantification of number of plaques (Fig. ), average plaque size (Fig. ), or distribution of small, medium, or large plaques (Fig. ), revealed no changes with treatment. Fig. 6 Lower-dose CSF1R inhibition does not alter Aβ or Tau levels. a, b Sandwich ELISA in soluble and insoluble fraction revealed no significant differences in Aβ40 or Aβ42 between groups. c, d Thioflavin-S staining was performed, revealing plaques in both control and treated groups. Of the subiculum, 10 × images are shown. e - g Analysis of plaques revealed no significant differe..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Lower-dose PLX5622 treatment prevents microglial association with plaques",
"text": "However, stark differences were seen in the microglia that are associated with plaques-in vehicle-treated animals, microglia were densely packed around plaques and displayed an activated morphology (Fig. ). Mice treated with PLX5622 showed comparable plaque load, but microglia were not associated with plaques to the same extent (Fig. ), suggesting that low doses of PLX5622 do not fully eliminate microglia but alter their response to inflammatory stimuli, such as plaques. Indeed, closeup z-stacks showed a clear association of microglia with plaques in untreated 3xTg-AD mice (Fig. ) but a lack of association in PLX5622-treated mice (Fig. ). Quantification of the number of microglia associated with a plaque and normalized to plaque diameter revealed a 70 % reduction in microglia associated with plaques. To explore the possibility that this lower dose of..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Lower-dose PLX5622 treatment prevents microglial association with plaques",
"text": "3xTg-AD mice also show progressive tau pathology as they age, including somatodendritic accumulation of human tau and hyperphosphorylation [ ]. Quantification of total human tau accumulation within somatodendritic compartments of CA1 neurons showed no differences with treatment, nor tau phosphorylated at S202/T205 (Fig. )."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice",
"datePublished": "2015",
"author": [
{
"@type": "Person",
"name": "Nabil N. Dagher"
},
{
"@type": "Person",
"name": "Allison R. Najafi"
},
{
"@type": "Person",
"name": "Kara M. Neely Kayala"
},
{
"@type": "Person",
"name": "Monica R. P. Elmore"
},
{
"@type": "Person",
"name": "Terra E. White"
},
{
"@type": "Person",
"name": "Rodrigo Medeiros"
},
{
"@type": "Person",
"name": "Brian L. West"
},
{
"@type": "Person",
"name": "Kim N. Green"
}
],
"identifier": "10.1186/s12974-015-0366-9"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice methods",
"item": "https://replicatescience.com/experiments/colony-stimulating-factor-1-receptor-inhibition-prevents-microglial-plaque-association-and-improves-cognition-in-3xtg-ad-mice-methods-nabil-n-dagher-pmc4522109/colony-stimulating-factor-1-receptor-inhibition-prevents-microglial-plaque-association-and-improves--mlph0ed4"
}
]
}
]