TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke methods
Aim. Evidence-backed execution summary for TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke methods from TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To determine which cell type is the source of increased TGFβ production after stroke, brain sections were stained with an anti-TGFβ a...
Western blotting
reagent used in the protocol.
- Use
- Unstroked and stroked brains were lysed in glo lysis buffer (Promega). Protein concentrations were equalized and lysates mixed with 4 × NuPage LDS loading buffer (Invitrogen). Samples were loaded onto 4-12% NuPage Bis-Tris gels (Invitrogen) and subsequently transferred onto PVDF membranes. Blots were incubated w...
TGFβ signaling increases with age and stroke
reagent used in the protocol.
- Use
- To confirm the increase in TGFβ signaling we detected by luciferase activity, we performed a western blot on brain extracts from the ipsilateral hemisphere of mice sacrificed before, and 7 days after stroke, for phosphorylated Smad2 (pSmad2), a downstream mediator of TGFβ signaling. The pSmad2 antibody we...
TGF-β1 is predominantly co-localized with CD68+ cells after stroke
reagent used in the protocol.
- Use
- To determine which cell type is the likely source of increased TGF-β1 production after stroke, brain sections were stained with an anti-TGF-β1 antibody, colocalized with markers for astrocytes (GFAP), neurons (Milli-mark Pan-Neuronal marker), and monocytic immune cells (CD68). Although TGF-β1 colocali...
TGFβ signaling in neurons, oligodendrocytes and endothelial cells is unchanged by stroke
reagent used in the protocol.
- Use
- TGFβ signaling in neurons, oligodendrocytes and endothelial cells is unchanged by stroke. A.) Co-localization of pSmad2 (marker of TGFβ signaling) with Millimark Pan-Neuronal antibody cocktail (marker of neurons) See additional file for a 3D projection. B.) Co-localization of pSmad2 with CAII (oligodendro...
Bioluminescent imaging
Bioluminescence was detected as described previously using the In Vivo Imaging System (IVIS; Xenogen, Alameda, CA) [ ]. Mice were injected intraperitoneally with 150 mg/kg D-luciferin (Xenogen) 10 min before imaging and anesthetized with isoflurane during imaging. Photons emitted from living mice were acquired as ph...
- Use
- Bioluminescence was detected as described previously using the In Vivo Imaging System (IVIS; Xenogen, Alameda, CA) [ ]. Mice were injected intraperitoneally with 150 mg/kg D-luciferin (Xenogen) 10 min before imaging and anesthetized with isoflurane during imaging. Photons emitted from living mice were acquired as ph...
Statistical analysis
Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (GraphPad, San Diego, CA). Means between two groups were compared with two-tailed, unpaired Student's t tests; comparisons of means from mult...
- Use
- Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (GraphPad, San Diego, CA). Means between two groups were compared with two-tailed, unpaired Student's t tests; comparisons of means from mult...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (GraphPad, San Diego, CA). Means between two groups were compared with two-tailed, unpaired Student's t tests; comparisons of means from mult...
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Methods
SBE-LucRT mice were a gift from Dr Tony Wyss-Coray [ ]. Nine females and 5 males (5 months old) were used to gauge variability in bioluminescence between individual mice, and to determine if bioluminescence is affected by the estrus cycle. Eighteen young females (5 months old) and eighteen old females (18 months old) were used to measure TGFβ signaling after stroke in real time by bioluminescence. 3 mice from each cohort were sacrificed at days 1, 3, 7, 14 and 21 after stroke for localization of TGF-β1 and TGFβ signaling by immunoflorescence. Three mice of each age group also underwent sham surgery and were imaged for 21 days prior to sacrifice for localization of TGFβ signaling in uninjured brains. This experimental paradigm was repeated in young and old male mice.
Surgery
Distal middle cerebral artery occlusion (dMCAO) was induced as described previously [ ]. Briefly, mice were anesthetized by isoflurane inhalation (2% isoflurane in 100% oxygen) and the skin over the right temple was shaved. Skin was swabbed with chlorehexidine and an incision was made to expose the temporalis muscle. A pocket was created in the temporalis muscle to expose the skull underneath and the right middle cerebral artery (MCA) was identified. A microdrill was used to penetrate the skull and expose the underlying MCA. The meninges were cut and the vessel was ligated using a small vessel cauterizer. The temporalis muscle was replaced and the wound closed using surgical glue. Throughout surgery body temperature was maintained at 37°C using a feedback controlled heating blanket.
Bioluminescent imaging
Bioluminescence was detected as described previously using the In Vivo Imaging System (IVIS; Xenogen, Alameda, CA) [ ]. Mice were injected intraperitoneally with 150 mg/kg D-luciferin (Xenogen) 10 min before imaging and anesthetized with isoflurane during imaging. Photons emitted from living mice were acquired as photons per second per cm 2 per steradian (sr) by using LIVINGIMAGE software (Xenogen) and integrated over 5 min. For photon quantification, a region of interest was manually selected and kept constant within all experiments. Baseline imaging was performed 1 week before stroke was induced and bioluminescence was expressed as fold induction over baseline levels.
Infarct evaluation
To visualize the region of infarction, representative animals from each age group (n ≥ 5) were sacrificed at 24 hours post stroke and their brains processed for TTC staining. Brains were sectioned into 1 mm thick slices and each slice was immersed in 1.5% TTC in PBS at 37°C for 15 minutes and then fixed in 10% formalin. The area of the ipsilateral hemisphere and the area of the infarct on each section was measured in a blinded fashion using NIH ImageJ 1.43u. The measurements were multiplied by the distance between sections (1 mm) and then summed over the entire brain to yield volume measurements.
TGFβ signaling increases with age and stroke
The dMCAO model of stroke produces a small lesion in the cortex with a clearly defined border and a small penumbra. In line with previous reports infarct volume was significantly larger in the aged mice and astrogliosis (GFAP immunoreactivity) was accelerated (Fig ) [, ]. Bioluminescence in the 5 month old females was increased 2 fold on day 1 post stroke, remained elevated for 7 days post stroke and was localized to the region of the infarct (Fig ). After 7 days TGFβ signaling began to return to baseline (Fig ). This fold increase was similar in the 18 month old female mice (Fig ), although the absolute amount of TGFβ signaling in the old mice was substantially higher (Fig ). This is not surprising considering that the lesion was much larger in the aged animals. However, TGFβ signaling was also significantly higher in the old animals at baseline, demonstrating that in...
TGFβ signaling increases with age and stroke
TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantification of GFAP immunostain, showing that astrogliosis is accelerated in aged mice (*p < 0.05 compared to 5 month old). C.) Representative bioluminescence showing reporter gene (luciferase) expression is increased after stroke compared to surgical shams (2 females shown at 3 days post stroke). D.) Time course of TGFβ signaling (luciferase induction) after stroke in 5 month old females (*p < 0.05 compared to sham). E.) Time course of TGFβ signaling (luciferase induction) after stroke in 18 month old females (*p < 0.05 compared to sham). F.) Comparison of absolute values of bioluminescence between young and old mice, showing that TGFβ signaling in the brain increases at baseline and after stroke (*...
TGFβ signaling increases with age and stroke
To confirm the increase in TGFβ signaling we detected by luciferase activity, we performed a western blot on brain extracts from the ipsilateral hemisphere of mice sacrificed before, and 7 days after stroke, for phosphorylated Smad2 (pSmad2), a downstream mediator of TGFβ signaling. The pSmad2 antibody we used for immunoflorescence (Millipore AB3849) detected a protein with the correct molecular weight for pSmad2 (58 kDa) and bound the same band as an alternative pSmad2 antibody (Cell Signaling 3101; Fig ). This verifies that the pSmad2 antibody we used for subsequent immunoflorescence is specific for pSmad2. In accordance with our luciferase findings pSmad2 levels were increased 7 days post stroke (Fig ).
TGFβ signaling in neurons, oligodendrocytes and endothelial cells is unchanged by stroke
To determine which cells are responding to TGFβ in the uninjured brain and after stroke, brain sections from mice sacrificed at days 1, 3, 7, 14 and 21 after stroke and at 21 days after sham surgery were double-labeled with anti-pSmad2, a marker of TGFβ signaling, and markers of neurons (Millimark Pan Neuronal marker), oligodendrocytes (CAII), endothelial cells (β-dystroglycan), astrocytes (GFAP) and activated microglia and macrophages (CD68). We found that neurons uniformly respond to TGFβ in the absence of injury and at all time points after stroke, with 100% of neurons co-localizing with pSmad2 (Fig and Fig; see additional file to confirm colocalization by 3D reconstruction). We also found that the majority (approximately 70%) of oligodendrocytes consistently respond to TGFβ in the absence of injury and did not observe any differences after stroke (Fig and...
Measurement outputs
What raw and processed outputs should exist?
We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To...
- 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
Unstroked and stroked brains were lysed in glo lysis buffer (Promega). Protein concentrations were equalized and lysates mixed with 4 × NuPage LDS loading buffer (Invitrogen...
- 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
Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (...
- 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
TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantif...
- 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
Data was acquired in a blinded and unbiased fashion.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To...; Unstroked and stroked brains were lysed in glo lysis buffer (Promega). Protein concentrations were equalized and lysates mixed with 4 × NuPage LDS loading buffer (Invitrogen...; Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (...; TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantif....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (...; TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantif...
from paperReporting output
Report representative outputs alongside summary comparisons for We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To..., Unstroked and stroked brains were lysed in glo lysis buffer (Promega). Protein concentrations were equalized and lysates mixed with 4 × NuPage LDS loading buffer (Invitrogen..., Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (..., TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantif....
inferred from protocolStructured statistical methods
Data was acquired in a blinded and unbiased fashion. Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed with Prism 5 software (...; TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantif...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
SBE-LucRT mice were a gift from Dr Tony Wyss-Coray [ ]. Nine females and 5 males (5 months old) were used to gauge variability in bioluminescence between individual mice, and to determine if bioluminescence is affected by the estrus cycle. Eighteen young females (5 months old) and eighteen old females (18 months old) were used to measure TGFβ signaling after stroke in real time by bioluminescence. 3 mice from each cohort were sacrificed at days 1, 3, 7, 14 and 21 after stroke for localization of TGF-β1 and TGFβ signaling by immunoflorescence. Three mice of each age group also underwent sham surgery and were imaged for 21 days prior to sacrifice for localization of TGFβ signaling in uninjured brains. This experimental paradigm was repeated in young and old male mice.
Distal middle cerebral artery occlusion (dMCAO) was induced as described previously [ ]. Briefly, mice were anesthetized by isoflurane inhalation (2% isoflurane in 100% oxygen) and the skin over the right temple was shaved. Skin was swabbed with chlorehexidine and an incision was made to expose the temporalis muscle. A pocket was created in the temporalis muscle to expose the skull underneath and the right middle cerebral artery (MCA) was identified. A microdrill was used to penetrate the skull and expose the underlying MCA. The meninges were cut and the vessel was ligated using a small vessel cauterizer. The temporalis muscle was replaced and the wound closed using surgical glue. Throughout surgery body temperature was maintained at 37°C using a feedback controlled heating blanket.
Bioluminescence was detected as described previously using the In Vivo Imaging System (IVIS; Xenogen, Alameda, CA) [ ]. Mice were injected intraperitoneally with 150 mg/kg D-luciferin (Xenogen) 10 min before imaging and anesthetized with isoflurane during imaging. Photons emitted from living mice were acquired as photons per second per cm 2 per steradian (sr) by using LIVINGIMAGE software (Xenogen) and integrated over 5 min. For photon quantification, a region of interest was manually selected and kept constant within all experiments. Baseline imaging was performed 1 week before stroke was induced and bioluminescence was expressed as fold induction over baseline levels.
To visualize the region of infarction, representative animals from each age group (n ≥ 5) were sacrificed at 24 hours post stroke and their brains processed for TTC staining. Brains were sectioned into 1 mm thick slices and each slice was immersed in 1.5% TTC in PBS at 37°C for 15 minutes and then fixed in 10% formalin. The area of the ipsilateral hemisphere and the area of the infarct on each section was measured in a blinded fashion using NIH ImageJ 1.43u. The measurements were multiplied by the distance between sections (1 mm) and then summed over the entire brain to yield volume measurements.
The dMCAO model of stroke produces a small lesion in the cortex with a clearly defined border and a small penumbra. In line with previous reports infarct volume was significantly larger in the aged mice and astrogliosis (GFAP immunoreactivity) was accelerated (Fig ) [, ]. Bioluminescence in the 5 month old females was increased 2 fold on day 1 post stroke, remained elevated for 7 days post stroke and was localized to the region of the infarct (Fig ). After 7 days TGFβ signaling began to return to baseline (Fig ). This fold increase was similar in the 18 month old female mice (Fig ), although the absolute amount of TGFβ signaling in the old mice was substantially higher (Fig ). This is not surprising considering that the lesion was much larger in the aged animals. However, TGFβ signaling was also significantly higher in the old animals at baseline, demonstrating that in the absence of injury TGFβ signaling in the brain increases with age.
TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantification of GFAP immunostain, showing that astrogliosis is accelerated in aged mice (*p < 0.05 compared to 5 month old). C.) Representative bioluminescence showing reporter gene (luciferase) expression is increased after stroke compared to surgical shams (2 females shown at 3 days post stroke). D.) Time course of TGFβ signaling (luciferase induction) after stroke in 5 month old females (*p < 0.05 compared to sham). E.) Time course of TGFβ signaling (luciferase induction) after stroke in 18 month old females (*p < 0.05 compared to sham). F.) Comparison of absolute values of bioluminescence between young and old mice, showing that TGFβ signaling in the brain increases at baseline and after stroke (*p < 0.05 compared to 5 month old). G.) Western blot to corroborate that TGFβ signalling increases after stroke. Two different antibodies for pSmad2 were used to confirm that pSmad2 is increased at day 7 after stroke. NS = no stroke, D7 = day 7 post stroke. All error bars for this figure are SEM.
To confirm the increase in TGFβ signaling we detected by luciferase activity, we performed a western blot on brain extracts from the ipsilateral hemisphere of mice sacrificed before, and 7 days after stroke, for phosphorylated Smad2 (pSmad2), a downstream mediator of TGFβ signaling. The pSmad2 antibody we used for immunoflorescence (Millipore AB3849) detected a protein with the correct molecular weight for pSmad2 (58 kDa) and bound the same band as an alternative pSmad2 antibody (Cell Signaling 3101; Fig ). This verifies that the pSmad2 antibody we used for subsequent immunoflorescence is specific for pSmad2. In accordance with our luciferase findings pSmad2 levels were increased 7 days post stroke (Fig ).
To determine which cells are responding to TGFβ in the uninjured brain and after stroke, brain sections from mice sacrificed at days 1, 3, 7, 14 and 21 after stroke and at 21 days after sham surgery were double-labeled with anti-pSmad2, a marker of TGFβ signaling, and markers of neurons (Millimark Pan Neuronal marker), oligodendrocytes (CAII), endothelial cells (β-dystroglycan), astrocytes (GFAP) and activated microglia and macrophages (CD68). We found that neurons uniformly respond to TGFβ in the absence of injury and at all time points after stroke, with 100% of neurons co-localizing with pSmad2 (Fig and Fig; see additional file to confirm colocalization by 3D reconstruction). We also found that the majority (approximately 70%) of oligodendrocytes consistently respond to TGFβ in the absence of injury and did not observe any differences after stroke (Fig and Fig; see additional file to confirm colocalization by 3D reconstruction). There was no qualitative difference in the intensity of colocalization of pSmad2 with neurons or oliogdendrocytes, at any time point after stroke. This data suggests that the response of neurons and oligodendrocytes to TGF&#...
Machine-readable layer
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"text": "Distal middle cerebral artery occlusion (dMCAO) was induced as described previously [ ]. Briefly, mice were anesthetized by isoflurane inhalation (2% isoflurane in 100% oxygen) and the skin over the right temple was shaved. Skin was swabbed with chlorehexidine and an incision was made to expose the temporalis muscle. A pocket was created in the temporalis muscle to expose the skull underneath and the right middle cerebral artery (MCA) was identified. A microdrill was used to penetrate the skull and expose the underlying MCA. The meninges were cut and the vessel was ligated using a small vessel cauterizer. The temporalis muscle was replaced and the wound closed using surgical glue. Throughout surgery body temperature was maintained at 37°C using a feedback controlled heating blanket."
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"text": "The dMCAO model of stroke produces a small lesion in the cortex with a clearly defined border and a small penumbra. In line with previous reports infarct volume was significantly larger in the aged mice and astrogliosis (GFAP immunoreactivity) was accelerated (Fig ) [, ]. Bioluminescence in the 5 month old females was increased 2 fold on day 1 post stroke, remained elevated for 7 days post stroke and was localized to the region of the infarct (Fig ). After 7 days TGFβ signaling began to return to baseline (Fig ). This fold increase was similar in the 18 month old female mice (Fig ), although the absolute amount of TGFβ signaling in the old mice was substantially higher (Fig ). This is not surprising considering that the lesion was much larger in the aged animals. However, TGFβ signaling was also significantly higher in the old animals at baseline, demonstrating that in..."
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"text": "TGFβ signaling in the brain increases with age and stroke. A.) Infarct volume and representative TTC images of the lesion 24 hours following dCMAO (*p < 0.05). B.) Quantification of GFAP immunostain, showing that astrogliosis is accelerated in aged mice (*p < 0.05 compared to 5 month old). C.) Representative bioluminescence showing reporter gene (luciferase) expression is increased after stroke compared to surgical shams (2 females shown at 3 days post stroke). D.) Time course of TGFβ signaling (luciferase induction) after stroke in 5 month old females (*p < 0.05 compared to sham). E.) Time course of TGFβ signaling (luciferase induction) after stroke in 18 month old females (*p < 0.05 compared to sham). F.) Comparison of absolute values of bioluminescence between young and old mice, showing that TGFβ signaling in the brain increases at baseline and after stroke (*..."
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"text": "To determine which cells are responding to TGFβ in the uninjured brain and after stroke, brain sections from mice sacrificed at days 1, 3, 7, 14 and 21 after stroke and at 21 days after sham surgery were double-labeled with anti-pSmad2, a marker of TGFβ signaling, and markers of neurons (Millimark Pan Neuronal marker), oligodendrocytes (CAII), endothelial cells (β-dystroglycan), astrocytes (GFAP) and activated microglia and macrophages (CD68). We found that neurons uniformly respond to TGFβ in the absence of injury and at all time points after stroke, with 100% of neurons co-localizing with pSmad2 (Fig and Fig; see additional file to confirm colocalization by 3D reconstruction). We also found that the majority (approximately 70%) of oligodendrocytes consistently respond to TGFβ in the absence of injury and did not observe any differences after stroke (Fig and..."
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