Pericyte loss influences Alzheimer-like neurodegeneration in mice methods
Aim. Evidence-backed execution summary for Pericyte loss influences Alzheimer-like neurodegeneration in mice methods from Pericyte loss influences Alzheimer-like neurodegeneration in mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Pericytes control Aβ clearance in APP mice
reagent used in the protocol.
- Use
- APP sw/0; Pdgfrβ +/- mice did not show changes in brain microvascular expression of other known Aβ transporters such as P-glycoprotein and receptor for advanced age glycation products ( ) or changes in the levels of Aβ-degrading enzymes in the brain-that is, insulin-degrading enzyme and n...
Measurement of sAPP-β levels and β-secretase activity
reagent used in the protocol.
- Use
- sAPP-β levels in the brain of APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice were measured using the ELISA kit (Covance) and β-secretase activity was determined using a β-secretase activity kit (Abcam).
Tissue immunofluorescent and fluorescent thioflavin-S and lectin staining
reagent used in the protocol.
- Use
- Mice were anesthetized as described above and transcardially perfused with phosphate buffered saline (PBS) containing 5 U ml -1 heparin. Brains were dissected and embedded into optimal cutting temperature compound (Tissue-Tek, Torrance, CA, USA) on dry ice. Optimal cutting temperature-embedded froz...
Bright field microscopy analysis
reagent used in the protocol.
- Use
- Mice were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS. Brains were postfixed in 4% paraformaldehyde overnight at 4 °C and embedded in paraplast. Serial sections were cut at 5 µm using a microtome, mounted on glass slides and rehydrated according to the standard protocols...
Aβ40- and Aβ42-specific ELISA
reagent used in the protocol.
- Use
- Cortex and hippocampus were dissected and homogenized in ice-cold guanidine buffer (5 M guanidine hydrochloride/50 mM TrisCl, pH 8.0). Aβ40 and Aβ42 levels were determined in samples using human-specific ELISA kits (Invitrogen) according to the manufacturer's instructions. For human A^...
Western blotting
reagent used in the protocol.
- Use
- All samples were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 1.0% NP-40, 0.5% sodium deoxycholate and Roche Applied Science, Indianapolis, IN, USA, protease inhibitor cocktail). Samples were then subjected to SDS-PAGE gel electrophoresis and transferred to a nitrocellulose memb...
In vivo microdialysis and ISF Aβ half-life determination
reagent used in the protocol.
- Use
- In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West L...
CY3-Aβ40 uptake and internalization by pericytes
reagent used in the protocol.
- Use
- To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3-4 h (ref. ). The cells were then spun down and washe...
Multiphoton imaging
One day before imaging animals received an intraperitoneal injection of 10 mg kg -1 methoxy-X04 (Neuroptix, Acton, MA, USA). The following day the mice were anesthetized using initially 5% isoflurane, and then within 15-30 s mice were placed on a heating pad (37 °C) and maint...
- Use
- One day before imaging animals received an intraperitoneal injection of 10 mg kg -1 methoxy-X04 (Neuroptix, Acton, MA, USA). The following day the mice were anesthetized using initially 5% isoflurane, and then within 15-30 s mice were placed on a heating pad (37 °C) and maint...
Confocal microscopy
All images were taken with a Zeiss 510 confocal microscopy and analysed using the NIH Image J software. Briefly, the number of CD13-positive pericytes and NeuN-positive neurons were analysed using the Image J cell counter tool and expressed per mm 2 of total tissue area. The length of capillaries (≤6 &#...
- Use
- All images were taken with a Zeiss 510 confocal microscopy and analysed using the NIH Image J software. Briefly, the number of CD13-positive pericytes and NeuN-positive neurons were analysed using the Image J cell counter tool and expressed per mm 2 of total tissue area. The length of capillaries (≤6 &#...
Bright field microscopy analysis
Mice were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS. Brains were postfixed in 4% paraformaldehyde overnight at 4 °C and embedded in paraplast. Serial sections were cut at 5 µm using a microtome, mounted on glass slides and rehydrated according to the standard protocols...
- Use
- Mice were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS. Brains were postfixed in 4% paraformaldehyde overnight at 4 °C and embedded in paraplast. Serial sections were cut at 5 µm using a microtome, mounted on glass slides and rehydrated according to the standard protocols...
In vivo microdialysis and ISF Aβ half-life determination
In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West L...
- Use
- In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West L...
CY3-Aβ40 uptake and internalization by pericytes
To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3-4 h (ref. ). The cells were then spun down and washe...
- Use
- To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3-4 h (ref. ). The cells were then spun down and washe...
Cell death in Aβ-treated pericyte cultures
Brain murine pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific). Cells were cultured for 7 days in the presence and absence of 5 µM Aβ40. Medium with and without 5 µM Aβ40 was replaced after 3 days. Cell viability was quantified using a fluorescent...
- Use
- Brain murine pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific). Cells were cultured for 7 days in the presence and absence of 5 µM Aβ40. Medium with and without 5 µM Aβ40 was replaced after 3 days. Cell viability was quantified using a fluorescent...
Voltage-sensitive dye imaging of cortical activity
For VSD imaging, a cranial window was placed over the somatosensory cortex using the same procedure as for Multiphoton imaging above. After removing the dura, without causing any cranial bleeding, RH-1692 VSD (Optical Imaging), dissolved in aCSF was applied to the exposed cortex. The brain was washed with aCSF for...
- Use
- For VSD imaging, a cranial window was placed over the somatosensory cortex using the same procedure as for Multiphoton imaging above. After removing the dura, without causing any cranial bleeding, RH-1692 VSD (Optical Imaging), dissolved in aCSF was applied to the exposed cortex. The brain was washed with aCSF for...
Laser doppler flowmetry
Cerebral blood flow responses to vibrissal stimulation in anesthetized 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice (750 mg kg -1 urethane and 50 mg kg -1 chloralose) were determined with laser Doppler flowmetry. The tip of the laser Doppler...
- Use
- Cerebral blood flow responses to vibrissal stimulation in anesthetized 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice (750 mg kg -1 urethane and 50 mg kg -1 chloralose) were determined with laser Doppler flowmetry. The tip of the laser Doppler...
In vivo microdialysis and ISF Aβ half-life determination
Software used for acquisition, scoring, statistics, or reporting.
- Use
- In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West L...
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Pericytes control Aβ clearance in APP mice
We next show that primary cultured murine brain pericytes rapidly clear extracellular Cy3-labelled Aβ40 via low-density lipoprotein receptor-related protein 1 (LRP1), a key Aβ clearance receptor in brain vasculature, that is normally expressed in brain pericytes in vivo ( ). LRP1-mediated Aβ clearance by cultured pericytes has been demonstrated by administering antibodies to block the function of specific LDL receptors ( ) and by quantifying the effects of silencing different LDL receptors with specific siRNA-blocking agents ( ). Moreover, adenoviral-mediated re-expression of human LRP1 minigene rescued the ability of pericytes with siRNA-induced LRP1 knockdown to clear Aβ ( ). Excessive LRP1-mediated accumulation of Aβ in pericytes over longer periods of time such as 7 days resulted in cell death ( ) similar to as reported in human brain pericyte cultures....
Tissue immunofluorescent and fluorescent thioflavin-S and lectin staining
Mice were anesthetized as described above and transcardially perfused with phosphate buffered saline (PBS) containing 5 U ml -1 heparin. Brains were dissected and embedded into optimal cutting temperature compound (Tissue-Tek, Torrance, CA, USA) on dry ice. Optimal cutting temperature-embedded frozen brain tissue was cryosectioned at a thickness of 14-18 µm and subsequently fixed in ice-cold acetone. Sections were blocked with 5% normal swine serum (Vector Laboratories, Burlingame, CA, USA) for 60 min and incubated in primary antibody diluted in blocking solution overnight at 4 °C. We used the following primary antibodies: rabbit anti-human Aβ (Cell Signaling Technology; no. 8243; 1:200), mouse anti-mouse Aβ (Invitrogen; AMB0062; 1:200), goat anti-mouse PDGFRβ (R&D Systems; AF1042; 1:100), mouse anti-NeuN (Millipore; M...
In vivo microdialysis and ISF Aβ half-life determination
In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West Lafayette, IN, USA) was stereotaxically implanted into the left hippocampus of the mouse (coordinates: AP -3.1 mm, L +2.4 mm and DV -0.6 mm at a 12° angle). A small head block (Instech laboratories, Plymouth Meeting, PA, USA) that provides tether anchoring to the freely moving system was attached to the skull. The cannula and the head block were cemented into place using dental acrylic. The microdialysis probes had a 2-mm, 38-kDa molecular weight cutoff membrane (Bioanalytical Systems) and were washed with 4% bovine serum albumin -artificia...
CY3-Aβ40 uptake and internalization by pericytes
To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3-4 h (ref. ). The cells were then spun down and washed with PBS and plated in a complete medium containing Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum, 1% non-essential amino acids, 1% vitamins and 1% antibiotic/antimycotic on plastic (non-coated) tissue culture plates. After 6-12 h, the non-adherent cells were rinsed away and fresh medium was replaced every 2-3 days. Cultures were confirmed to be morphologically consistent with pericyte cultures and were PDGFRβ-positive, desmin-positive, glial fibrillar acidic protein-negative, aquaporin 4-negative, microtubule-associated prot...
Cell death in Aβ-treated pericyte cultures
Brain murine pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific). Cells were cultured for 7 days in the presence and absence of 5 µM Aβ40. Medium with and without 5 µM Aβ40 was replaced after 3 days. Cell viability was quantified using a fluorescent Live/Dead Viability/Cytotoxicity kit according to the manufacturer's instruction (Invitrogen). In some experiments, cells were treated with anti-LRP1 antibody or si Lrp1 for 7 days, as described above. Images were obtained using an inverted microscope (DMI6000B, Leica Microsystems Inc., Buffalo Grove, IL, USA). Data were analysed with the NIH ImageJ counter tool.
Behavioural testing
For the novel object recognition test, mice were acclimatized to a 25-cm 3 cubic box for 10 min, and then exposed at three-time-point trials to two objects affixed to the floor, equidistant to the two nearest walls. Mice were placed in a corner equidistant to both objects, minimizing spatial memory confounds. Starting position for mice was rotated, and objects counterbalanced throughout the test. All trials were videotaped for 5 min. Baseline trial was performed 24 h after acclimatization with two identical objects. A choice trial was performed 90 min after baseline, replacing one familiar object with a novel object and keeping one baseline object constant. Total duration of exploratory approaches to familiar or novel objects was measured and was defined as sniffing or touching an object with the snout at a critical distance of <1 cm from object. The nov...
Voltage-sensitive dye imaging of cortical activity
For VSD imaging, a cranial window was placed over the somatosensory cortex using the same procedure as for Multiphoton imaging above. After removing the dura, without causing any cranial bleeding, RH-1692 VSD (Optical Imaging), dissolved in aCSF was applied to the exposed cortex. The brain was washed with aCSF for 5 min, covered with low-melt agarose dissolved in aCSF (~1.3%), sealed with a coverslip and the skull was secured to a custom-built microscopy frame. Images were collected using a Pixelfly CCD camera coupled to the CamWare 3.0 software. RH-1692 was excited using a 627-nm LED light source and imaged using a custom-built Olympus 2 × epifluorescent microscope. Images were collected for 500 ms before and after a mechanical deflection of the hindlimb 5 ms in duration. The responses were averaged from 10-20 trials per animal. Stimulation trial signals...
Measurement outputs
What raw and processed outputs should exist?
We next show that primary cultured murine brain pericytes rapidly clear extracellular Cy3-labelled Aβ40 via low-density lipoprotein receptor-related protein 1 (LRP1), a key...
- 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
The relative levels of LRP1 expression in brain microvessels and pericytes isolated from APP sw/0; Pdgfrβ +/- mice and APP sw/0; Pdgfrβ +/+ littermate controls...
- 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
APP sw/0; Pdgfrβ +/- mice did not show changes in brain microvascular expression of other known Aβ transporters such as P-glycoprotein and receptor for advanced...
- 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
Next, we studied whether pericyte loss can influence the development of tau pathology and neurodegenerative changes in APP sw/0 mice. Our immunocytochemical analysis in APP sw/0...
- 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 determine whether pericyte deficiency affects Aβ clearance, brain interstitial fluid (ISF) Aβ levels were monitored by hippocampal in vivo microdialysis in 3-4-month-old pericyte-deficient APP sw/0; Pdgfrβ +/- mice with a 31% loss of pericytes in...
from paperScoring or quantification
Quantify the primary readouts for this experiment: We next show that primary cultured murine brain pericytes rapidly clear extracellular Cy3-labelled Aβ40 via low-density lipoprotein receptor-related protein 1 (LRP1), a key...; The relative levels of LRP1 expression in brain microvessels and pericytes isolated from APP sw/0; Pdgfrβ +/- mice and APP sw/0; Pdgfrβ +/+ littermate controls...; APP sw/0; Pdgfrβ +/- mice did not show changes in brain microvascular expression of other known Aβ transporters such as P-glycoprotein and receptor for advanced...; Next, we studied whether pericyte loss can influence the development of tau pathology and neurodegenerative changes in APP sw/0 mice. Our immunocytochemical analysis in APP sw/0....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
To determine whether pericyte deficiency affects Aβ clearance, brain interstitial fluid (ISF) Aβ levels were monitored by hippocampal in vivo microdialysis in 3-...; APP sw/0 mice develop high plasma Aβ levels, raising a possibility that plasma Aβ may contribute and increase brain Aβ levels by their transport across the blood&...; Next, we studied whether pericyte loss can influence the development of tau pathology and neurodegenerative changes in APP sw/0 mice. Our immunocytochemical analysis in APP sw/0...; Importantly, pericyte deficiency led to progressive neuronal degenerative changes as evidenced by ~50% reductions in the neurite density and 23-25% loss of neurons in the...
from paperReporting output
Report representative outputs alongside summary comparisons for We next show that primary cultured murine brain pericytes rapidly clear extracellular Cy3-labelled Aβ40 via low-density lipoprotein receptor-related protein 1 (LRP1), a key..., The relative levels of LRP1 expression in brain microvessels and pericytes isolated from APP sw/0; Pdgfrβ +/- mice and APP sw/0; Pdgfrβ +/+ littermate controls..., APP sw/0; Pdgfrβ +/- mice did not show changes in brain microvascular expression of other known Aβ transporters such as P-glycoprotein and receptor for advanced..., Next, we studied whether pericyte loss can influence the development of tau pathology and neurodegenerative changes in APP sw/0 mice. Our immunocytochemical analysis in APP sw/0....
inferred from protocolStructured statistical methods
To determine whether pericyte deficiency affects Aβ clearance, brain interstitial fluid (ISF) Aβ levels were monitored by hippocampal in vivo microdialysis in 3-...; APP sw/0 mice develop high plasma Aβ levels, raising a possibility that plasma Aβ may contribute and increase brain Aβ levels by their transport across the blood&...; Next, we studied whether pericyte loss can influence the development of tau pathology and neurodegenerative changes in APP sw/0 mice. Our immunocytochemical analysis in APP sw/0...; Importantly, pericyte deficiency led to progressive neuronal degenerative changes as evidenced by ~50% reductions in the neurite density and 23-25% loss of neurons in the...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
We next show that primary cultured murine brain pericytes rapidly clear extracellular Cy3-labelled Aβ40 via low-density lipoprotein receptor-related protein 1 (LRP1), a key Aβ clearance receptor in brain vasculature, that is normally expressed in brain pericytes in vivo ( ). LRP1-mediated Aβ clearance by cultured pericytes has been demonstrated by administering antibodies to block the function of specific LDL receptors ( ) and by quantifying the effects of silencing different LDL receptors with specific siRNA-blocking agents ( ). Moreover, adenoviral-mediated re-expression of human LRP1 minigene rescued the ability of pericytes with siRNA-induced LRP1 knockdown to clear Aβ ( ). Excessive LRP1-mediated accumulation of Aβ in pericytes over longer periods of time such as 7 days resulted in cell death ( ) similar to as reported in human brain pericyte cultures. These findings not only suggest that Aβ clearance by pericytes is critical for Aβ homeostasis but also show that extreme Aβ accumulation in pericytes leads to cell death. We next show in vivo that Aβ-overproducing APP sw/0 mice have an age-dependent loss of pericytes from 17% at...
Mice were anesthetized as described above and transcardially perfused with phosphate buffered saline (PBS) containing 5 U ml -1 heparin. Brains were dissected and embedded into optimal cutting temperature compound (Tissue-Tek, Torrance, CA, USA) on dry ice. Optimal cutting temperature-embedded frozen brain tissue was cryosectioned at a thickness of 14-18 µm and subsequently fixed in ice-cold acetone. Sections were blocked with 5% normal swine serum (Vector Laboratories, Burlingame, CA, USA) for 60 min and incubated in primary antibody diluted in blocking solution overnight at 4 °C. We used the following primary antibodies: rabbit anti-human Aβ (Cell Signaling Technology; no. 8243; 1:200), mouse anti-mouse Aβ (Invitrogen; AMB0062; 1:200), goat anti-mouse PDGFRβ (R&D Systems; AF1042; 1:100), mouse anti-NeuN (Millipore; MAB377; 1:200), mouse anti-neurofilament SMI-311 (Abcam; ab24575; 1:1,000) and goat anti-CD13 (R&D Systems; AF2335; 1:200). Sections were washed in PBS and incubated with the following secondary antibodies: Alexa 488-conjugated donkey anti-rabbit (Invitrogen; A11008; 1:200) to detect human Aβ, A...
In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West Lafayette, IN, USA) was stereotaxically implanted into the left hippocampus of the mouse (coordinates: AP -3.1 mm, L +2.4 mm and DV -0.6 mm at a 12° angle). A small head block (Instech laboratories, Plymouth Meeting, PA, USA) that provides tether anchoring to the freely moving system was attached to the skull. The cannula and the head block were cemented into place using dental acrylic. The microdialysis probes had a 2-mm, 38-kDa molecular weight cutoff membrane (Bioanalytical Systems) and were washed with 4% bovine serum albumin -artificial cerebrospinal fluid (aCSF) (Harvard Apparatus, Holliston, MA, USA) before use. After implantation of the guide cannula, the stylet was removed and the microdialysis probe inserted through the guide cannula into the hippocampus. The tethering system connected to a swivel (Instech) and counter balan...
To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3-4 h (ref. ). The cells were then spun down and washed with PBS and plated in a complete medium containing Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum, 1% non-essential amino acids, 1% vitamins and 1% antibiotic/antimycotic on plastic (non-coated) tissue culture plates. After 6-12 h, the non-adherent cells were rinsed away and fresh medium was replaced every 2-3 days. Cultures were confirmed to be morphologically consistent with pericyte cultures and were PDGFRβ-positive, desmin-positive, glial fibrillar acidic protein-negative, aquaporin 4-negative, microtubule-associated protein 2-negative, NeuN-negative, von Willebrand Factor-negative and ionized calcium-binding adapter molecule 1-negative. Primary pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific) and grown overnight. For Cy3-Aβ40 uptake experiments, pericytes were initially i...
Brain murine pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific). Cells were cultured for 7 days in the presence and absence of 5 µM Aβ40. Medium with and without 5 µM Aβ40 was replaced after 3 days. Cell viability was quantified using a fluorescent Live/Dead Viability/Cytotoxicity kit according to the manufacturer's instruction (Invitrogen). In some experiments, cells were treated with anti-LRP1 antibody or si Lrp1 for 7 days, as described above. Images were obtained using an inverted microscope (DMI6000B, Leica Microsystems Inc., Buffalo Grove, IL, USA). Data were analysed with the NIH ImageJ counter tool.
For the novel object recognition test, mice were acclimatized to a 25-cm 3 cubic box for 10 min, and then exposed at three-time-point trials to two objects affixed to the floor, equidistant to the two nearest walls. Mice were placed in a corner equidistant to both objects, minimizing spatial memory confounds. Starting position for mice was rotated, and objects counterbalanced throughout the test. All trials were videotaped for 5 min. Baseline trial was performed 24 h after acclimatization with two identical objects. A choice trial was performed 90 min after baseline, replacing one familiar object with a novel object and keeping one baseline object constant. Total duration of exploratory approaches to familiar or novel objects was measured and was defined as sniffing or touching an object with the snout at a critical distance of <1 cm from object. The novelty exploration index was calculated as time spent exploring the novel object over total time exploring both objects.
For VSD imaging, a cranial window was placed over the somatosensory cortex using the same procedure as for Multiphoton imaging above. After removing the dura, without causing any cranial bleeding, RH-1692 VSD (Optical Imaging), dissolved in aCSF was applied to the exposed cortex. The brain was washed with aCSF for 5 min, covered with low-melt agarose dissolved in aCSF (~1.3%), sealed with a coverslip and the skull was secured to a custom-built microscopy frame. Images were collected using a Pixelfly CCD camera coupled to the CamWare 3.0 software. RH-1692 was excited using a 627-nm LED light source and imaged using a custom-built Olympus 2 × epifluorescent microscope. Images were collected for 500 ms before and after a mechanical deflection of the hindlimb 5 ms in duration. The responses were averaged from 10-20 trials per animal. Stimulation trial signals were divided by baseline signal profiles collected in the absence of stimulation. The signal intensity was quantified by placing a circular region area of interest over the hindlimb region using the NIH Image software. The change in fluorescent intensity (ΔF/F 0 ) was calculated as a percent c...
Machine-readable layer
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"name": "Pericyte loss influences Alzheimer-like neurodegeneration in mice methods",
"description": "Evidence-backed execution summary for Pericyte loss influences Alzheimer-like neurodegeneration in mice methods from Pericyte loss influences Alzheimer-like neurodegeneration in mice.",
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"name": "Pericytes control Aβ clearance in APP mice",
"text": "We next show that primary cultured murine brain pericytes rapidly clear extracellular Cy3-labelled Aβ40 via low-density lipoprotein receptor-related protein 1 (LRP1), a key Aβ clearance receptor in brain vasculature, that is normally expressed in brain pericytes in vivo ( ). LRP1-mediated Aβ clearance by cultured pericytes has been demonstrated by administering antibodies to block the function of specific LDL receptors ( ) and by quantifying the effects of silencing different LDL receptors with specific siRNA-blocking agents ( ). Moreover, adenoviral-mediated re-expression of human LRP1 minigene rescued the ability of pericytes with siRNA-induced LRP1 knockdown to clear Aβ ( ). Excessive LRP1-mediated accumulation of Aβ in pericytes over longer periods of time such as 7 days resulted in cell death ( ) similar to as reported in human brain pericyte cultures...."
},
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"name": "Tissue immunofluorescent and fluorescent thioflavin-S and lectin staining",
"text": "Mice were anesthetized as described above and transcardially perfused with phosphate buffered saline (PBS) containing 5 U ml -1 heparin. Brains were dissected and embedded into optimal cutting temperature compound (Tissue-Tek, Torrance, CA, USA) on dry ice. Optimal cutting temperature-embedded frozen brain tissue was cryosectioned at a thickness of 14-18 µm and subsequently fixed in ice-cold acetone. Sections were blocked with 5% normal swine serum (Vector Laboratories, Burlingame, CA, USA) for 60 min and incubated in primary antibody diluted in blocking solution overnight at 4 °C. We used the following primary antibodies: rabbit anti-human Aβ (Cell Signaling Technology; no. 8243; 1:200), mouse anti-mouse Aβ (Invitrogen; AMB0062; 1:200), goat anti-mouse PDGFRβ (R&D Systems; AF1042; 1:100), mouse anti-NeuN (Millipore; M..."
},
{
"@type": "HowToStep",
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"name": "In vivo microdialysis and ISF Aβ half-life determination",
"text": "In vivo microdialysis was used to measure soluble Aβ40 and Aβ42 steady-state levels in the hippocampus of awake, freely moving 3- to 4-month-old APP sw/0 Pdgfrβ +/+ and APP sw/0 Pdgfrβ +/- mice. Under isoflurane anesthetic, an intracerebral guide cannula MRB-5 (Bioanalytical Systems, West Lafayette, IN, USA) was stereotaxically implanted into the left hippocampus of the mouse (coordinates: AP -3.1 mm, L +2.4 mm and DV -0.6 mm at a 12° angle). A small head block (Instech laboratories, Plymouth Meeting, PA, USA) that provides tether anchoring to the freely moving system was attached to the skull. The cannula and the head block were cemented into place using dental acrylic. The microdialysis probes had a 2-mm, 38-kDa molecular weight cutoff membrane (Bioanalytical Systems) and were washed with 4% bovine serum albumin -artificia..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "CY3-Aβ40 uptake and internalization by pericytes",
"text": "To isolate brain murine pericytes, isolated microvessel fragments from mouse cortex and hippocampus were digested for 12 h at 37 °C with collagenase A (Roche Applied Science), followed by constant shaking and vigorous pipetting every 3-4 h (ref. ). The cells were then spun down and washed with PBS and plated in a complete medium containing Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum, 1% non-essential amino acids, 1% vitamins and 1% antibiotic/antimycotic on plastic (non-coated) tissue culture plates. After 6-12 h, the non-adherent cells were rinsed away and fresh medium was replaced every 2-3 days. Cultures were confirmed to be morphologically consistent with pericyte cultures and were PDGFRβ-positive, desmin-positive, glial fibrillar acidic protein-negative, aquaporin 4-negative, microtubule-associated prot..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Cell death in Aβ-treated pericyte cultures",
"text": "Brain murine pericytes were plated into an eight-well chambered coverglass (Nunc, Thermo Scientific). Cells were cultured for 7 days in the presence and absence of 5 µM Aβ40. Medium with and without 5 µM Aβ40 was replaced after 3 days. Cell viability was quantified using a fluorescent Live/Dead Viability/Cytotoxicity kit according to the manufacturer's instruction (Invitrogen). In some experiments, cells were treated with anti-LRP1 antibody or si Lrp1 for 7 days, as described above. Images were obtained using an inverted microscope (DMI6000B, Leica Microsystems Inc., Buffalo Grove, IL, USA). Data were analysed with the NIH ImageJ counter tool."
},
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"position": 6,
"name": "Behavioural testing",
"text": "For the novel object recognition test, mice were acclimatized to a 25-cm 3 cubic box for 10 min, and then exposed at three-time-point trials to two objects affixed to the floor, equidistant to the two nearest walls. Mice were placed in a corner equidistant to both objects, minimizing spatial memory confounds. Starting position for mice was rotated, and objects counterbalanced throughout the test. All trials were videotaped for 5 min. Baseline trial was performed 24 h after acclimatization with two identical objects. A choice trial was performed 90 min after baseline, replacing one familiar object with a novel object and keeping one baseline object constant. Total duration of exploratory approaches to familiar or novel objects was measured and was defined as sniffing or touching an object with the snout at a critical distance of <1 cm from object. The nov..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Voltage-sensitive dye imaging of cortical activity",
"text": "For VSD imaging, a cranial window was placed over the somatosensory cortex using the same procedure as for Multiphoton imaging above. After removing the dura, without causing any cranial bleeding, RH-1692 VSD (Optical Imaging), dissolved in aCSF was applied to the exposed cortex. The brain was washed with aCSF for 5 min, covered with low-melt agarose dissolved in aCSF (~1.3%), sealed with a coverslip and the skull was secured to a custom-built microscopy frame. Images were collected using a Pixelfly CCD camera coupled to the CamWare 3.0 software. RH-1692 was excited using a 627-nm LED light source and imaged using a custom-built Olympus 2 × epifluorescent microscope. Images were collected for 500 ms before and after a mechanical deflection of the hindlimb 5 ms in duration. The responses were averaged from 10-20 trials per animal. Stimulation trial signals..."
}
],
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{
"@type": "HowToTool",
"name": "Multiphoton imaging"
},
{
"@type": "HowToTool",
"name": "Confocal microscopy"
},
{
"@type": "HowToTool",
"name": "Bright field microscopy analysis"
},
{
"@type": "HowToTool",
"name": "In vivo microdialysis and ISF Aβ half-life determination"
},
{
"@type": "HowToTool",
"name": "CY3-Aβ40 uptake and internalization by pericytes"
},
{
"@type": "HowToTool",
"name": "Cell death in Aβ-treated pericyte cultures"
},
{
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"name": "Voltage-sensitive dye imaging of cortical activity"
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{
"@type": "HowToTool",
"name": "Laser doppler flowmetry"
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{
"@type": "HowToSupply",
"name": "Pericytes control Aβ clearance in APP mice"
},
{
"@type": "HowToSupply",
"name": "Measurement of sAPP-β levels and β-secretase activity"
},
{
"@type": "HowToSupply",
"name": "Tissue immunofluorescent and fluorescent thioflavin-S and lectin staining"
},
{
"@type": "HowToSupply",
"name": "Bright field microscopy analysis"
},
{
"@type": "HowToSupply",
"name": "Aβ40- and Aβ42-specific ELISA"
},
{
"@type": "HowToSupply",
"name": "Western blotting"
},
{
"@type": "HowToSupply",
"name": "In vivo microdialysis and ISF Aβ half-life determination"
},
{
"@type": "HowToSupply",
"name": "CY3-Aβ40 uptake and internalization by pericytes"
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"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Pericyte loss influences Alzheimer-like neurodegeneration in mice",
"datePublished": "2013",
"author": [
{
"@type": "Person",
"name": "Abhay P. Sagare"
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{
"@type": "Person",
"name": "Robert D. Bell"
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{
"@type": "Person",
"name": "Zhen Zhao"
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{
"@type": "Person",
"name": "Qingyi Ma"
},
{
"@type": "Person",
"name": "Ethan A. Winkler"
},
{
"@type": "Person",
"name": "Anita Ramanathan"
},
{
"@type": "Person",
"name": "Berislav V. Zlokovic"
}
],
"identifier": "10.1038/ncomms3932"
}
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