Microglia regulate central nervous system myelin growth and integrity methods
Aim. Evidence-backed execution summary for Microglia regulate central nervous system myelin growth and integrity methods from Microglia regulate central nervous system myelin growth and integrity.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Microglia depletion in adulthood
reagent used in the protocol.
- Use
- The CSF1R inhibitor PLX5622 (Chemgood, C-1521) was formulated into chow at a concentration of 1,200 ppm (Research Diets, D11100404i) and fed to 2-month-old and 5-month-old wild-type ( Fire +/+ ) mice for 1 month and euthanized as described above.
Microglia prevent demyelination
reagent used in the protocol.
- Use
- Assessment of myelin in Fire Δ/Δ mice at 6 months of age showed areas of substantial demyelination (Fig. ) and areas of patchy demyelination. This in turn resulted in a significant decrease in the number and proportion of myelinated axons compared with Fire +/+ mice (Fig. ). The patchy nature of demy...
Microglia maintain existing myelin
reagent used in the protocol.
- Use
- We next asked whether these changes in myelin growth and integrity reflect disruption of myelin formation or myelin maintenance. To that end, we depleted microglia in mice when developmental myelination is complete (2 months of age onwards) by providing the CSF1R inhibitor PLX5622 in the diet of adult Fire +/+...
Electron microscopy of mouse tissue
reagent used in the protocol.
- Use
- Mice were intracardially perfused with 4% PFA (w/v) and 2% glutaraldehyde (v/v; TAAB Laboratories) in 0.1 M phosphate buffer. Tissue was post-fixed overnight at 4 °C and transferred to 1% glutaraldehyde (v/v) until embedding. Tissue sections (1 mm) were post-fixed in 1% osmium tetroxide and de...
Immunofluorescence staining of mouse tissue
reagent used in the protocol.
- Use
- Mice were intracardially perfused with 4% paraformaldehyde (PFA; w/v; Sigma), and brains were post-fixed overnight and cryoprotected in sucrose before embedding in OCT (Tissue-Tech) and storage at -80 °C. Cryosections (10 µm) were air-dried, permeabilized and blocked for 1 h with 5%...
Flow cytometry
reagent used in the protocol.
- Use
- An enzyme-free brain dissociation protocol was used to gather a myeloid cell-enriched cell suspension to explore CSF1R (CD115) surface protein levels. After transcardially perfusing 10-11-week-old female mice with ice-cold PBS, brains were dissected and minced with a 22A scalpel in HBSS (without Ca 2+ and Mg 2...
Measuring TGFβ1 levels by ELISA
reagent used in the protocol.
- Use
- Following perfusion with PBS, the corpus callosum was dissected from 2 mm coronal sections of Fire Δ/Δ and wild-type brains and snap-frozen in liquid nitrogen. Samples were homogenized in RIPA buffer (Millipore, 20-188) containing phosphatase and protease inhibitors (Sigma-Aldrich, 4906845001 a...
Brain dissociation and cell sorting for single-cell RNA sequencing
reagent used in the protocol.
- Use
- Brains were collected from 6-7-week-old female mice at the same time of day for each animal. Mice were culled by cervical dislocation and brains were dissected, with the olfactory bulbs and cerebellum removed. Hippocampi from both hemispheres and the remainder of the left hemisphere (without the hippocampus) w...
Main
Myelin ensheathes neuronal axons to ensure their health and rapid propagation of electrical impulses to support central nervous system (CNS) functions, for example, cognition. Learning and memory involve the formation of myelin and require myelin to be of good structural integrity. Myelin layers are compacted to a t...
- Use
- Myelin ensheathes neuronal axons to ensure their health and rapid propagation of electrical impulses to support central nervous system (CNS) functions, for example, cognition. Learning and memory involve the formation of myelin and require myelin to be of good structural integrity. Myelin layers are compacted to a t...
Microglia prevent hypermyelination
Given that these changes in myelin structure are sufficient to cause cognitive impairment in other models,, we evaluated cognition in Fire Δ/Δ mice using the Barnes maze spatial learning and memory task (Extended Data Fig. ). Both Fire Δ/Δ and Fire +/+ mice became progressively faster at locati...
- Use
- Given that these changes in myelin structure are sufficient to cause cognitive impairment in other models,, we evaluated cognition in Fire Δ/Δ mice using the Barnes maze spatial learning and memory task (Extended Data Fig. ). Both Fire Δ/Δ and Fire +/+ mice became progressively faster at locati...
Microglia suppress oligodendrocyte state
We next sought to determine the cellular and molecular mechanisms that underpin the loss of myelin health in the absence of microglia. To that end, we performed single-cell RNA sequencing of brain samples from Fire Δ/Δ and Fire +/+ mice at 1 month of age (Extended Data Fig. ). Mature oligodendrocytes...
- Use
- We next sought to determine the cellular and molecular mechanisms that underpin the loss of myelin health in the absence of microglia. To that end, we performed single-cell RNA sequencing of brain samples from Fire Δ/Δ and Fire +/+ mice at 1 month of age (Extended Data Fig. ). Mature oligodendrocytes...
Electron microscopy of mouse tissue
Mice were intracardially perfused with 4% PFA (w/v) and 2% glutaraldehyde (v/v; TAAB Laboratories) in 0.1 M phosphate buffer. Tissue was post-fixed overnight at 4 °C and transferred to 1% glutaraldehyde (v/v) until embedding. Tissue sections (1 mm) were post-fixed in 1% osmium tetroxide and de...
- Use
- Mice were intracardially perfused with 4% PFA (w/v) and 2% glutaraldehyde (v/v; TAAB Laboratories) in 0.1 M phosphate buffer. Tissue was post-fixed overnight at 4 °C and transferred to 1% glutaraldehyde (v/v) until embedding. Tissue sections (1 mm) were post-fixed in 1% osmium tetroxide and de...
Behavioural testing
Experimenters were blinded to genotype during behavioural testing and data analyses. All experiments were performed in a behaviour testing room maintained at a constant temperature of 20 °C. The open-field test was performed on male mice at 4-8 weeks and 11-13 months of age to asse...
- Use
- Experimenters were blinded to genotype during behavioural testing and data analyses. All experiments were performed in a behaviour testing room maintained at a constant temperature of 20 °C. The open-field test was performed on male mice at 4-8 weeks and 11-13 months of age to asse...
Methods
All experiments were performed under project licences approved by the UK Home Office and issued under the Animals (Scientific Procedures) Act. This study used Csf1r Fire Δ/Δ mice on a mixed CBA:C57Bl6J background, wild-type controls from Csf1r Fire Δ/+ crossings, Plp creERT mice (Jackson Laboratories)...
- Use
- All experiments were performed under project licences approved by the UK Home Office and issued under the Animals (Scientific Procedures) Act. This study used Csf1r Fire Δ/Δ mice on a mixed CBA:C57Bl6J background, wild-type controls from Csf1r Fire Δ/+ crossings, Plp creERT mice (Jackson Laboratories)...
Genotyping
Genomic DNA was extracted from ear biopsy tissue using a Wizard SV genomic purification system (Promega) according to the manufacturer's instructions. Csf1r Fire Δ/Δ mice were genotyped using PCR strategies as previously described. Genotyping of Plp creERT;Tgfbr1 fl/fl mice was performed with genom...
- Use
- Genomic DNA was extracted from ear biopsy tissue using a Wizard SV genomic purification system (Promega) according to the manufacturer's instructions. Csf1r Fire Δ/Δ mice were genotyped using PCR strategies as previously described. Genotyping of Plp creERT;Tgfbr1 fl/fl mice was performed with genom...
Immunofluorescence staining of mouse tissue
Mice were intracardially perfused with 4% paraformaldehyde (PFA; w/v; Sigma), and brains were post-fixed overnight and cryoprotected in sucrose before embedding in OCT (Tissue-Tech) and storage at -80 °C. Cryosections (10 µm) were air-dried, permeabilized and blocked for 1 h with 5%...
- Use
- Mice were intracardially perfused with 4% paraformaldehyde (PFA; w/v; Sigma), and brains were post-fixed overnight and cryoprotected in sucrose before embedding in OCT (Tissue-Tech) and storage at -80 °C. Cryosections (10 µm) were air-dried, permeabilized and blocked for 1 h with 5%...
Statistics and reproducibility
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All manual cell counts were performed in a blinded manner. Data are presented as the mean ± s.e.m. All micrographs are representative images of the result, which were independently repeated a minimum of three times; exact n values are presented in the corresponding graphs of quantification. Before s...
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SRI-011381 hydrochloride administration
The TGFβ signalling agonist SRI-011381 hydrochloride (HY-100347A, Cambridge Bioscience/MedChem Express), dissolved in PBS containing DMSO (10%) and PEG300 (40%), was injected intraperitoneally into Fire Δ/Δ mice at a dose of 30 mg kg -1 3 times per week for 3 weeks (9 injections in total) and killed as described above.
Behavioural testing
Experimenters were blinded to genotype during behavioural testing and data analyses. All experiments were performed in a behaviour testing room maintained at a constant temperature of 20 °C. The open-field test was performed on male mice at 4-8 weeks and 11-13 months of age to assess locomotor activity and anxiety-associated behaviours. Handling was carried out 3-4 days before testing. Mice were placed in the open field (47 × 47 cm) to freely explore the arena for 10 min. Equipment was cleaned with 70% ethanol between each test to remove odours. The total ambulatory distance travelled (in metres) and the time spent in the edges (9 cm from the wall) and centre (29 × 29 cm) were automatically quantified using the video tracking software Any-Maze (Stoelting Europe, v.6.3). The Barnes...
Immunofluorescence staining of mouse tissue
Mice were intracardially perfused with 4% paraformaldehyde (PFA; w/v; Sigma), and brains were post-fixed overnight and cryoprotected in sucrose before embedding in OCT (Tissue-Tech) and storage at -80 °C. Cryosections (10 µm) were air-dried, permeabilized and blocked for 1 h with 5% normal horse serum (Gibco) and 0.3% Triton-X-100 (Fisher Scientific) in PBS. For myelin protein staining, sections were permeabilized in methanol at -20 °C for 10 min. For EdU visualization, an AlexaFluor-555 Click-iT EdU Cell Proliferation Assay kit (Invitrogen) was applied before immunostaining. Sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature (20-25 °C) then incubated in Click-iT reaction cocktail in the dark at room temperature for 30 min and then washed in PBS. Heat-induc...
Measuring TGFβ1 levels by ELISA
Following perfusion with PBS, the corpus callosum was dissected from 2 mm coronal sections of Fire Δ/Δ and wild-type brains and snap-frozen in liquid nitrogen. Samples were homogenized in RIPA buffer (Millipore, 20-188) containing phosphatase and protease inhibitors (Sigma-Aldrich, 4906845001 and 11836170001). Corpus callosum lysate samples were activated and TGFβ1 protein levels were measured by ELISA (BioLegend, 436707) according to the manufacturer's instructions for serum and plasma samples at a final dilution of 1:10. BCA assays were performed to measure total protein according to the manufacturer's instructions (Thermo Fisher Scientific, 23225), and values were normalized to this for each sample.
Lipidomics
Corpus callosum samples from Fire +/+ and Fire Δ/Δ mice were diluted in 700 µl of PBS with 800 µl 1 N HCl:CH 3 OH 1:8 (v/v), 900 µl ChCl 3 and 200 µg ml -1 of the antioxidant 2,6- di -tert-butyl-4-methylphenol (BHT; Sigma-Aldrich). Splash Lipidomix Mass Spec standard (3 µl; Avanti Polar Lipids) was added into the extract mix. The organic fraction was evaporated at room temperature using a Savant Speedvac spd111v (Thermo Fisher), and the remaining lipid pellet was reconstituted in 100% ethanol. Lipid species were analysed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX). Chromatographic separation was performed on...
Immunofluorescence staining of human tissue
Post-mortem tissue from individuals with ALSP and from unaffected individuals who had died of non-neurological causes (Extended Data Table ) were obtained with full ethical approval from the Queen Square Brain Bank for Neurological Disorders, UCL Queen Square Institute of Neurology, and their use was in accord with the terms of the informed consents for use of donor tissue and information. Ethical approval was granted to the Queen Square Brain Bank for the use of tissue by the National Health Service Health Research Authority through the London Central Research Ethics Committee. Diagnosis of ALSP was confirmed on the basis of mutations in CSF1R and by neuropathological means, and clinical history was provided by Z. Jaunmuktane (University College London). Formalin-fixed paraffin embedded tissue blocks were cut to 10 µm thickness. Sections were placed in the oven at 60...
Extended data figures and tables
Extended Data Fig. 4 Learning and memory encoding unimpaired, but cognitive flexibility is compromised, in the FIRE Δ/Δ mouse. a ) Training phase of Barnes maze: mice learn to locate Target hole 1 (orange) with underlying escape chamber in spatial learning and memory retrieval trials. Mice tested were 2-4 months of age, with a median age of 119 days old (FIRE +/+ ) and 120 days old (FIRE Δ/Δ ). b ) Mean primary latency (sec) ± s.e.m. over 6 training days. n = 13 FIRE +/+ mice and 10 FIRE Δ/Δ. Non-significant between genotypes across all training days, Day 1: ** P = 0.0011; Day 2: P = 0.9787; Day 3: P = 0.9627; Day 4: P = 0.9574; Day 5: P = 0.9990; Day 6: P > 0.9999, Repeated measures 2-way ANOVA with Sidak's multiple comparisons test. c ) Mean prima...
Extended data figures and tables
Extended Data Fig. 5 Post-learning oligodendrogenesis and myelination. a ) Representative images of EdU (magenta) and OLIG2 (white) double positive cells (arrows), with magnified view of dotted outline. Scale bars, 25 µm. b ) Mean OLIG2+ EdU+ cells/mm 2 ± s.e.m. n = 9 FIRE +/+ mice and 4 FIRE Δ/Δ mice. Non-significant, P = 0.9696, 2-tailed unpaired Student's t-test. c ) Mean proportion of OLIG2+ EdU+ cells which are mature oligodendrocytes (CC1+; black) or immature lineage cells (CC1-; grey) ± s.e.m. n = 9 FIRE +/+ mice and 4 FIRE Δ/Δ mice. Non-significant, CC1+: P > 0.9999; CC1-: P > 0.9999, 1-way ANOVA with Tukey's multiple comparisons test. d ) Representative images of EdU+ (magenta) CC1+ (green) OLIG2+ (white) triple positive cells (arrows). Scale bar, 25 µm....
Measurement outputs
What raw and processed outputs should exist?
To address these questions, we utilized a recently developed transgenic mouse model in which the Fms intronic regulatory element ( Fire ) super-enhancer of the Csf1r gene ( Fire...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
However, Fire Δ/Δ mice showed abnormal myelin structure indicative of hypermyelination. At P25-P30, there was an increase in myelin that was outfolding or unrave...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Given that these changes in myelin structure are sufficient to cause cognitive impairment in other models,, we evaluated cognition in Fire Δ/Δ mice using the Barnes...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
After demonstrating that microglia are required for myelin health in mice, we investigated the relevance of these findings in humans. We analysed samples from individuals with t...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
To address these questions, we utilized a recently developed transgenic mouse model in which the Fms intronic regulatory element ( Fire ) super-enhancer of the Csf1r gene ( Fire Δ/Δ; Fig.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To address these questions, we utilized a recently developed transgenic mouse model in which the Fms intronic regulatory element ( Fire ) super-enhancer of the Csf1r gene ( Fire...; However, Fire Δ/Δ mice showed abnormal myelin structure indicative of hypermyelination. At P25-P30, there was an increase in myelin that was outfolding or unrave...; Given that these changes in myelin structure are sufficient to cause cognitive impairment in other models,, we evaluated cognition in Fire Δ/Δ mice using the Barnes...; After demonstrating that microglia are required for myelin health in mice, we investigated the relevance of these findings in humans. We analysed samples from individuals with t....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
To address these questions, we utilized a recently developed transgenic mouse model in which the Fms intronic regulatory element ( Fire ) super-enhancer of the Csf1r gene ( Fire...; However, Fire Δ/Δ mice showed abnormal myelin structure indicative of hypermyelination. At P25-P30, there was an increase in myelin that was outfolding or unrave...; Assessment of myelin in Fire Δ/Δ mice at 6 months of age showed areas of substantial demyelination (Fig. ) and areas of patchy demyelination. This in turn result...; After demonstrating that microglia are required for myelin health in mice, we investigated the relevance of these findings in humans. We analysed samples from individuals with t...
from paperReporting output
Report representative outputs alongside summary comparisons for To address these questions, we utilized a recently developed transgenic mouse model in which the Fms intronic regulatory element ( Fire ) super-enhancer of the Csf1r gene ( Fire..., However, Fire Δ/Δ mice showed abnormal myelin structure indicative of hypermyelination. At P25-P30, there was an increase in myelin that was outfolding or unrave..., Given that these changes in myelin structure are sufficient to cause cognitive impairment in other models,, we evaluated cognition in Fire Δ/Δ mice using the Barnes..., After demonstrating that microglia are required for myelin health in mice, we investigated the relevance of these findings in humans. We analysed samples from individuals with t....
inferred from protocolStructured statistical methods
To address these questions, we utilized a recently developed transgenic mouse model in which the Fms intronic regulatory element ( Fire ) super-enhancer of the Csf1r gene ( Fire...; However, Fire Δ/Δ mice showed abnormal myelin structure indicative of hypermyelination. At P25-P30, there was an increase in myelin that was outfolding or unrave...; Assessment of myelin in Fire Δ/Δ mice at 6 months of age showed areas of substantial demyelination (Fig. ) and areas of patchy demyelination. This in turn result...; After demonstrating that microglia are required for myelin health in mice, we investigated the relevance of these findings in humans. We analysed samples from individuals with t...
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Evidence quotes (8)
The TGFβ signalling agonist SRI-011381 hydrochloride (HY-100347A, Cambridge Bioscience/MedChem Express), dissolved in PBS containing DMSO (10%) and PEG300 (40%), was injected intraperitoneally into Fire Δ/Δ mice at a dose of 30 mg kg -1 3 times per week for 3 weeks (9 injections in total) and killed as described above.
Experimenters were blinded to genotype during behavioural testing and data analyses. All experiments were performed in a behaviour testing room maintained at a constant temperature of 20 °C. The open-field test was performed on male mice at 4-8 weeks and 11-13 months of age to assess locomotor activity and anxiety-associated behaviours. Handling was carried out 3-4 days before testing. Mice were placed in the open field (47 × 47 cm) to freely explore the arena for 10 min. Equipment was cleaned with 70% ethanol between each test to remove odours. The total ambulatory distance travelled (in metres) and the time spent in the edges (9 cm from the wall) and centre (29 × 29 cm) were automatically quantified using the video tracking software Any-Maze (Stoelting Europe, v.6.3). The Barnes maze test was performed in adult mice 2-4 months of age to assess spatial learning, memory and cognitive flexibility. The maze consisted of one white circular platform with 20 circular holes around the outside edge, 91.5 cm in diameter and 115 cm in height (San Diego Instrume...
Mice were intracardially perfused with 4% paraformaldehyde (PFA; w/v; Sigma), and brains were post-fixed overnight and cryoprotected in sucrose before embedding in OCT (Tissue-Tech) and storage at -80 °C. Cryosections (10 µm) were air-dried, permeabilized and blocked for 1 h with 5% normal horse serum (Gibco) and 0.3% Triton-X-100 (Fisher Scientific) in PBS. For myelin protein staining, sections were permeabilized in methanol at -20 °C for 10 min. For EdU visualization, an AlexaFluor-555 Click-iT EdU Cell Proliferation Assay kit (Invitrogen) was applied before immunostaining. Sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature (20-25 °C) then incubated in Click-iT reaction cocktail in the dark at room temperature for 30 min and then washed in PBS. Heat-induced antigen retrieval was performed before primary antibody application. Primary antibodies were applied overnight at 4 °C in a humid chamber and included the following: MBP (AbD Serotec, 1:250; MCA409S, clone 12); MAG (Millipore, 1:100; MAB1567, clone 513); MOG (Millipore, 1:100; MAB5680,...
Following perfusion with PBS, the corpus callosum was dissected from 2 mm coronal sections of Fire Δ/Δ and wild-type brains and snap-frozen in liquid nitrogen. Samples were homogenized in RIPA buffer (Millipore, 20-188) containing phosphatase and protease inhibitors (Sigma-Aldrich, 4906845001 and 11836170001). Corpus callosum lysate samples were activated and TGFβ1 protein levels were measured by ELISA (BioLegend, 436707) according to the manufacturer's instructions for serum and plasma samples at a final dilution of 1:10. BCA assays were performed to measure total protein according to the manufacturer's instructions (Thermo Fisher Scientific, 23225), and values were normalized to this for each sample.
Corpus callosum samples from Fire +/+ and Fire Δ/Δ mice were diluted in 700 µl of PBS with 800 µl 1 N HCl:CH 3 OH 1:8 (v/v), 900 µl ChCl 3 and 200 µg ml -1 of the antioxidant 2,6- di -tert-butyl-4-methylphenol (BHT; Sigma-Aldrich). Splash Lipidomix Mass Spec standard (3 µl; Avanti Polar Lipids) was added into the extract mix. The organic fraction was evaporated at room temperature using a Savant Speedvac spd111v (Thermo Fisher), and the remaining lipid pellet was reconstituted in 100% ethanol. Lipid species were analysed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX). Chromatographic separation was performed on a XBridge amide column (150 × 4.6 mm, 3 × 5 µm; Waters) maintained at 35 °C using mobile phase A (1 mM ammonium acetate in water and acetonitrile 5:95 (v/v)) and mobile phase B (1 mM ammonium acetate in water and...
Post-mortem tissue from individuals with ALSP and from unaffected individuals who had died of non-neurological causes (Extended Data Table ) were obtained with full ethical approval from the Queen Square Brain Bank for Neurological Disorders, UCL Queen Square Institute of Neurology, and their use was in accord with the terms of the informed consents for use of donor tissue and information. Ethical approval was granted to the Queen Square Brain Bank for the use of tissue by the National Health Service Health Research Authority through the London Central Research Ethics Committee. Diagnosis of ALSP was confirmed on the basis of mutations in CSF1R and by neuropathological means, and clinical history was provided by Z. Jaunmuktane (University College London). Formalin-fixed paraffin embedded tissue blocks were cut to 10 µm thickness. Sections were placed in the oven at 60 °C for 10 min and deparaffinized by a series of washes in HistoClear (2 × 10 min) and ethanol (100% twice, 95%, 70%, 50%; 5 min each). Following washes in PBS, slides were placed in a pressure cooker in Vector unmasking solution for 20 min, cooled, washed once...
Extended Data Fig. 4 Learning and memory encoding unimpaired, but cognitive flexibility is compromised, in the FIRE Δ/Δ mouse. a ) Training phase of Barnes maze: mice learn to locate Target hole 1 (orange) with underlying escape chamber in spatial learning and memory retrieval trials. Mice tested were 2-4 months of age, with a median age of 119 days old (FIRE +/+ ) and 120 days old (FIRE Δ/Δ ). b ) Mean primary latency (sec) ± s.e.m. over 6 training days. n = 13 FIRE +/+ mice and 10 FIRE Δ/Δ. Non-significant between genotypes across all training days, Day 1: ** P = 0.0011; Day 2: P = 0.9787; Day 3: P = 0.9627; Day 4: P = 0.9574; Day 5: P = 0.9990; Day 6: P > 0.9999, Repeated measures 2-way ANOVA with Sidak's multiple comparisons test. c ) Mean primary errors ± s.e.m. during training phase. n = 13 FIRE +/+ mice and 10 FIRE Δ/Δ mice. Non-significant, Day 1: P = 0.9649; Day 2: P = 0.5968; Day 3: P = 0.8884; Day 4: P = 0.6028; Day 5: P = 0.9215; Day 6: P &#...
Extended Data Fig. 5 Post-learning oligodendrogenesis and myelination. a ) Representative images of EdU (magenta) and OLIG2 (white) double positive cells (arrows), with magnified view of dotted outline. Scale bars, 25 µm. b ) Mean OLIG2+ EdU+ cells/mm 2 ± s.e.m. n = 9 FIRE +/+ mice and 4 FIRE Δ/Δ mice. Non-significant, P = 0.9696, 2-tailed unpaired Student's t-test. c ) Mean proportion of OLIG2+ EdU+ cells which are mature oligodendrocytes (CC1+; black) or immature lineage cells (CC1-; grey) ± s.e.m. n = 9 FIRE +/+ mice and 4 FIRE Δ/Δ mice. Non-significant, CC1+: P > 0.9999; CC1-: P > 0.9999, 1-way ANOVA with Tukey's multiple comparisons test. d ) Representative images of EdU+ (magenta) CC1+ (green) OLIG2+ (white) triple positive cells (arrows). Scale bar, 25 µm. e ) Images of corpus callosum 6 weeks post cognitive testing. Scale bar, 1 µm. f ) Mean number of myelinated axons/mm 2 ± s.e.m. in untrained versus trained mice. n = 3 mice/group in untrained category, 4 trained FIRE +/+ mice and 6 trained FIRE Δ/Δ mice. FIRE...
Machine-readable layer
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"text": "The TGFβ signalling agonist SRI-011381 hydrochloride (HY-100347A, Cambridge Bioscience/MedChem Express), dissolved in PBS containing DMSO (10%) and PEG300 (40%), was injected intraperitoneally into Fire Δ/Δ mice at a dose of 30 mg kg -1 3 times per week for 3 weeks (9 injections in total) and killed as described above."
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"name": "Immunofluorescence staining of mouse tissue",
"text": "Mice were intracardially perfused with 4% paraformaldehyde (PFA; w/v; Sigma), and brains were post-fixed overnight and cryoprotected in sucrose before embedding in OCT (Tissue-Tech) and storage at -80 °C. Cryosections (10 µm) were air-dried, permeabilized and blocked for 1 h with 5% normal horse serum (Gibco) and 0.3% Triton-X-100 (Fisher Scientific) in PBS. For myelin protein staining, sections were permeabilized in methanol at -20 °C for 10 min. For EdU visualization, an AlexaFluor-555 Click-iT EdU Cell Proliferation Assay kit (Invitrogen) was applied before immunostaining. Sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature (20-25 °C) then incubated in Click-iT reaction cocktail in the dark at room temperature for 30 min and then washed in PBS. Heat-induc..."
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"name": "Measuring TGFβ1 levels by ELISA",
"text": "Following perfusion with PBS, the corpus callosum was dissected from 2 mm coronal sections of Fire Δ/Δ and wild-type brains and snap-frozen in liquid nitrogen. Samples were homogenized in RIPA buffer (Millipore, 20-188) containing phosphatase and protease inhibitors (Sigma-Aldrich, 4906845001 and 11836170001). Corpus callosum lysate samples were activated and TGFβ1 protein levels were measured by ELISA (BioLegend, 436707) according to the manufacturer's instructions for serum and plasma samples at a final dilution of 1:10. BCA assays were performed to measure total protein according to the manufacturer's instructions (Thermo Fisher Scientific, 23225), and values were normalized to this for each sample."
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"position": 5,
"name": "Lipidomics",
"text": "Corpus callosum samples from Fire +/+ and Fire Δ/Δ mice were diluted in 700 µl of PBS with 800 µl 1 N HCl:CH 3 OH 1:8 (v/v), 900 µl ChCl 3 and 200 µg ml -1 of the antioxidant 2,6- di -tert-butyl-4-methylphenol (BHT; Sigma-Aldrich). Splash Lipidomix Mass Spec standard (3 µl; Avanti Polar Lipids) was added into the extract mix. The organic fraction was evaporated at room temperature using a Savant Speedvac spd111v (Thermo Fisher), and the remaining lipid pellet was reconstituted in 100% ethanol. Lipid species were analysed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX). Chromatographic separation was performed on..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Immunofluorescence staining of human tissue",
"text": "Post-mortem tissue from individuals with ALSP and from unaffected individuals who had died of non-neurological causes (Extended Data Table ) were obtained with full ethical approval from the Queen Square Brain Bank for Neurological Disorders, UCL Queen Square Institute of Neurology, and their use was in accord with the terms of the informed consents for use of donor tissue and information. Ethical approval was granted to the Queen Square Brain Bank for the use of tissue by the National Health Service Health Research Authority through the London Central Research Ethics Committee. Diagnosis of ALSP was confirmed on the basis of mutations in CSF1R and by neuropathological means, and clinical history was provided by Z. Jaunmuktane (University College London). Formalin-fixed paraffin embedded tissue blocks were cut to 10 µm thickness. Sections were placed in the oven at 60..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Extended data figures and tables",
"text": "Extended Data Fig. 4 Learning and memory encoding unimpaired, but cognitive flexibility is compromised, in the FIRE Δ/Δ mouse. a ) Training phase of Barnes maze: mice learn to locate Target hole 1 (orange) with underlying escape chamber in spatial learning and memory retrieval trials. Mice tested were 2-4 months of age, with a median age of 119 days old (FIRE +/+ ) and 120 days old (FIRE Δ/Δ ). b ) Mean primary latency (sec) ± s.e.m. over 6 training days. n = 13 FIRE +/+ mice and 10 FIRE Δ/Δ. Non-significant between genotypes across all training days, Day 1: ** P = 0.0011; Day 2: P = 0.9787; Day 3: P = 0.9627; Day 4: P = 0.9574; Day 5: P = 0.9990; Day 6: P > 0.9999, Repeated measures 2-way ANOVA with Sidak's multiple comparisons test. c ) Mean prima..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Extended data figures and tables",
"text": "Extended Data Fig. 5 Post-learning oligodendrogenesis and myelination. a ) Representative images of EdU (magenta) and OLIG2 (white) double positive cells (arrows), with magnified view of dotted outline. Scale bars, 25 µm. b ) Mean OLIG2+ EdU+ cells/mm 2 ± s.e.m. n = 9 FIRE +/+ mice and 4 FIRE Δ/Δ mice. Non-significant, P = 0.9696, 2-tailed unpaired Student's t-test. c ) Mean proportion of OLIG2+ EdU+ cells which are mature oligodendrocytes (CC1+; black) or immature lineage cells (CC1-; grey) ± s.e.m. n = 9 FIRE +/+ mice and 4 FIRE Δ/Δ mice. Non-significant, CC1+: P > 0.9999; CC1-: P > 0.9999, 1-way ANOVA with Tukey's multiple comparisons test. d ) Representative images of EdU+ (magenta) CC1+ (green) OLIG2+ (white) triple positive cells (arrows). Scale bar, 25 µm...."
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"name": "Measuring TGFβ1 levels by ELISA"
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