Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice methods
Aim. Evidence-backed execution summary for Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice methods from Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice.
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mouse
Subject model for the experiment.
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Lymphatic outflow of a macromolecular tracer from CSF
reagent used in the protocol.
- Use
- Quantification of lymphatic outflow of CSF has been attempted in many species using approaches such as recovery of radiolabeled tracers through cannulation of collecting lymphatic vessels or detection of signal in draining lymph nodes with imaging approaches,,,. Our above findings have revealed that the multiple...
Lymphatic routes of CSF from the cranial cavity
We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 tracer near cranial nerve exit routes from the skull at 60 min after intraventricular infusion in Prox1-GFP mice,. Examination of the ba...
- Use
- We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 tracer near cranial nerve exit routes from the skull at 60 min after intraventricular infusion in Prox1-GFP mice,. Examination of the ba...
CSF outflow of small molecules through the lymphatic system
We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emission wavelength properties similar to P40D680 were utilized: Evans blue (EB), IRDye680CW, and a 3 kDa dextran conjugated to AlexaFluor...
- Use
- We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emission wavelength properties similar to P40D680 were utilized: Evans blue (EB), IRDye680CW, and a 3 kDa dextran conjugated to AlexaFluor...
Infusion of tracers into lateral ventricle or cisterna magna
Mice were anesthetized (80 mg/kg ketamine; 0.2 mg/kg medetomidine) and fixed in a stereotaxic frame (RWD, San Diego, CA). The skull was thinned with a dental drill (RWD) at a location 0.95 mm lateral and 0.22 mm caudal to the bregma. A 33 G steel needle was inserted into the right later...
- Use
- Mice were anesthetized (80 mg/kg ketamine; 0.2 mg/kg medetomidine) and fixed in a stereotaxic frame (RWD, San Diego, CA). The skull was thinned with a dental drill (RWD) at a location 0.95 mm lateral and 0.22 mm caudal to the bregma. A 33 G steel needle was inserted into the right later...
Dynamic NIR imaging of CSF outflow
For noninvasive imaging of tracer signals in blood, fur above the saphenous vein region was shaved with a razor and depilation cream before the ventricular or cisternal infusion. After removal of the needle from the skull or the cisterna magna, the mice were quickly positioned under a Zeiss StereoLumar.V12 stereomi...
- Use
- For noninvasive imaging of tracer signals in blood, fur above the saphenous vein region was shaved with a razor and depilation cream before the ventricular or cisternal infusion. After removal of the needle from the skull or the cisterna magna, the mice were quickly positioned under a Zeiss StereoLumar.V12 stereomi...
Assessment of transport to blood and lymph nodes
Using AxioVision software, a circular region of interest of radius 100 µm was placed over the saphenous vein on the acquired videos. Using the Measure Profile function, a table of fluorescence intensity in counts vs. time was exported into Microsoft Excel. As there was a loss of signal at the beginning of...
- Use
- Using AxioVision software, a circular region of interest of radius 100 µm was placed over the saphenous vein on the acquired videos. Using the Measure Profile function, a table of fluorescence intensity in counts vs. time was exported into Microsoft Excel. As there was a loss of signal at the beginning of...
Assessment of collecting lymphatic vessel contractility
Contractility was assessed based on our published methods. In short, in AxioVision software, on each video of dynamic imaging of mandibular lymph nodes, a region of interest of radius 200 µm was drawn over the afferent collecting vessel on each side of the mouse. The data in mean fluorescent intensity va...
- Use
- Contractility was assessed based on our published methods. In short, in AxioVision software, on each video of dynamic imaging of mandibular lymph nodes, a region of interest of radius 200 µm was drawn over the afferent collecting vessel on each side of the mouse. The data in mean fluorescent intensity va...
Intravenous infusions for tracer dose calibration
A custom-designed catheter of polyethylene (PE)-10 tubing (SCI, Lake Havasu City, AZ) and a 30 G needle was placed into the tail vein of C57BL/6 J mice. An infusion pump (PHD2000, Harvard Apparatus, Cambridge, MA) connected to the catheter was used for consistent infusions of tracer. For demonstrat...
- Use
- A custom-designed catheter of polyethylene (PE)-10 tubing (SCI, Lake Havasu City, AZ) and a 30 G needle was placed into the tail vein of C57BL/6 J mice. An infusion pump (PHD2000, Harvard Apparatus, Cambridge, MA) connected to the catheter was used for consistent infusions of tracer. For demonstrat...
In situ analysis of CSF flow and lymphatic outflow
Anatomical mapping of the outflow pathways in Prox1-GFP mice was performed in three groups of n = 5 mice at time points of euthanization at 10, 30, and 60 min after intraventricular infusion of P40D680. Images of P40D680 tracer spread on the surfaces of the brain, perineural exit points, and withi...
- Use
- Anatomical mapping of the outflow pathways in Prox1-GFP mice was performed in three groups of n = 5 mice at time points of euthanization at 10, 30, and 60 min after intraventricular infusion of P40D680. Images of P40D680 tracer spread on the surfaces of the brain, perineural exit points, and withi...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- For the studies to assess paravascular spread and CSF outflow at different time points, mice were randomly allocated to different experimental groups. Owing to the nature of the aging studies, neither randomization nor blinding of investigators was possible. Group sizes were estimated based on pilot studies to deter...
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Lymphatic routes of CSF from the cranial cavity
After euthanizing mice at 10, 30, and 60 min after infusion, we assessed the dynamics of the P40D680 signal within the deep cervical and mandibular lymph nodes of the neck. Deep cervical nodes already had signal at 10 min after the completion of the infusion, as shown in representative images of the left cervical nodes at this time point in Fig.. The signals steadily increased with time, with significantly more tracer found in the nodes at 60 min when compared to 10 min (25,916 ± 10,741 vs. 7723 ± 3333 counts, p < 0.05; one-way ANOVA, Fig. ). Although the mandibular lymph nodes had slightly delayed dynamics, all mice had tracer within the nodes at 30 min after infusion (Fig. ). A significantly increased amount of tracer was found at 60 min compared to 10 min after infusion (28,5...
Lymphatic routes of CSF from the cranial cavity
We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 tracer near cranial nerve exit routes from the skull at 60 min after intraventricular infusion in Prox1-GFP mice,. Examination of the base of the skull demonstrated accumulation of the tracer around the optic and trigeminal nerves (CN II and V) as they exit the skull (Fig. ). Tracer was also apparent at the cribriform plate that separates the cranial and nasal cavities, indicating that outflow had also occurred along the olfactory nerves (CN I). Removal of the skin around the eye allowed visualization of a dense plexus of tracer-filled lymphatic vessels exiting the orbit that coalesced into one collecting vessel coursing along the anterior facial vein towards the mandibular lymph nodes (Fig. )....
Lymphatic routes of CSF from the cranial cavity
CSF outflow pathways to deep cervical lymph nodes. a Representative image showing lymphatic outflow from the nasal cavity. Lower jaw, tongue, trachea, and esophagus have been removed. pal: palate, phx: pharynx. Inset shows a tracer-filled collecting lymphatic vessel. Scale bar: 2 mm. b Representative image showing outflow from the jugular foramen on the medial side of the tympanic bulla. n: cranial nerves IX, X, and XI, jv: jugular vein, ca: carotid artery. Scale bar: 500 µm. c Representative image showing the perineural outflow along the facial nerve (fn) from the stylomastoid foramen on the lateral side of the tympanic bulla towards a mandibular lymph node (m ln). Scale bar: 1 mm. Images are acquired at t = 60 min after infusion and are representative of n = 10 mice. d Scheme demonstrating the outflow routes in the deep cervical region. Gre...
Lymphatic outflow of a macromolecular tracer from CSF
Quantification of lymphatic outflow of CSF has been attempted in many species using approaches such as recovery of radiolabeled tracers through cannulation of collecting lymphatic vessels or detection of signal in draining lymph nodes with imaging approaches,,,. Our above findings have revealed that the multiple routes of lymphatic flow and the anatomical variation between animals indicate that such previous approaches would be limited. Therefore, we aimed to apply a technique recently developed in our lab, which allows quantification of lymphatic transport from an organ by measurement in a peripheral blood vessel of an interstitially injected tracer. This approach assumes that the tracer is lymphatic-specific for the tissue of interest and that the tracer is not retained in the tissue or within draining lymph nodes by phagocytosis. The P40D680 tracer that we have used in the ana...
Lymphatic outflow of a macromolecular tracer from CSF
We infused 2.5 µL of P40D680 into the right lateral ventricle and after 5 min initiated noninvasive imaging of the saphenous vein with NIR stereomicroscopy (Fig. ). The venous signal pattern demonstrated an approximate 25 min delay before signal was seen and a steady increase thereafter (Fig.; Supplementary Movie ). This pattern is indicative of lymph outflow as previously observed in the subcutaneous tissue and the peritoneal cavity. Unlike these tissues, however, outflow was robust from the CSF even under anesthesia, with an estimated 21.6% ± 6.0% ( n = 5 mice) of the infused dose within the bloodstream at 60 min based on the calibration curve shown in Supplementary Fig.. Fig. 5 Dynamics of CSF outflow to systemic blood and mandibular lymph nodes. a Representative GFP and P40D680 images of the saph...
CSF outflow of small molecules through the lymphatic system
We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emission wavelength properties similar to P40D680 were utilized: Evans blue (EB), IRDye680CW, and a 3 kDa dextran conjugated to AlexaFluor680 (3kDa-AF680). Initial experiments were performed to establish comparable doses for intraventricular infusion as well as to test the sensitivity of detection in the saphenous region. It was observed during these studies that although both IRDye680CW and 3kDa-AF680 were not retained within the blood compartment after intravenous injection, they leaked rapidly in sufficient quantities within the skin above the saphenous vein to be detected with high sensitivity (Supplementary Fig. ). In groups of n = 4 mice, we infused 2.5 µL of either 0.6% EB...
Reduced lymphatic outflow from CSF in aged mice
The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these disorders was proposed that toxic proteins such as amyloid beta and tau may accumulate in the brain due to reduced clearance. Earlier studies have shown a reduced turnover of CSF and removal of labeled proteins, including amyloid beta, after ventricular-cisternal perfusion in aged rats. Therefore, we tested whether lymphatic outflow from CSF was reduced in aged mice and would be similar to prior observations of reduced peripheral lymphatic transport in aged mice,,. We first tested whether we could detect differences in CSF transport to blood after ventricular infusion of P40D680 in 18-month-old (aged) and 2-month-old (young) C57BL/6J-Tyrc-J albino mice. After 60 min, we observed less P40D680 signal i...
Dynamic NIR imaging of CSF outflow
For noninvasive imaging of tracer signals in blood, fur above the saphenous vein region was shaved with a razor and depilation cream before the ventricular or cisternal infusion. After removal of the needle from the skull or the cisterna magna, the mice were quickly positioned under a Zeiss StereoLumar.V12 stereomicroscope with AxioVision software (Carl Zeiss, Feldbach, Switzerland) and a Photometrics Evolve 512 camera (Photometrics, Tuscon, AZ) in a supine position on a heating pad to maintain body temperature. The autofluorescence signal on the GFP channel was used to position the saphenous blood vessels at × 25 zoom. Dynamic imaging was initiated 5 min after the completion of the ventricle infusion and 15 min after completion of the cisterna infusion by acquisition of a sequence of images (1 image every 15 s for 55 min) with a Cy5 filter set to monitor...
Measurement outputs
What raw and processed outputs should exist?
We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 trac...
- 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
CSF outflow pathways to deep cervical lymph nodes. a Representative image showing lymphatic outflow from the nasal cavity. Lower jaw, tongue, trachea, and esophagus have been re...
- 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
We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emis...
- 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 incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these...
- 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
After euthanizing mice at 10, 30, and 60 min after infusion, we assessed the dynamics of the P40D680 signal within the deep cervical and mandibular lymph nodes of the neck.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 trac...; CSF outflow pathways to deep cervical lymph nodes. a Representative image showing lymphatic outflow from the nasal cavity. Lower jaw, tongue, trachea, and esophagus have been re...; We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emis...; The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
After euthanizing mice at 10, 30, and 60 min after infusion, we assessed the dynamics of the P40D680 signal within the deep cervical and mandibular lymph nodes of the neck...; We infused 2.5 µL of P40D680 into the right lateral ventricle and after 5 min initiated noninvasive imaging of the saphenous vein with NIR stereomicroscopy (Fig...; We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emis...; The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these...
from paperReporting output
Report representative outputs alongside summary comparisons for We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 trac..., CSF outflow pathways to deep cervical lymph nodes. a Representative image showing lymphatic outflow from the nasal cavity. Lower jaw, tongue, trachea, and esophagus have been re..., We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emis..., The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these....
inferred from protocolStructured statistical methods
After euthanizing mice at 10, 30, and 60 min after infusion, we assessed the dynamics of the P40D680 signal within the deep cervical and mandibular lymph nodes of the neck...; We infused 2.5 µL of P40D680 into the right lateral ventricle and after 5 min initiated noninvasive imaging of the saphenous vein with NIR stereomicroscopy (Fig...; We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emis...; The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these...
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What supports the facts on this page?
Evidence quotes (8)
After euthanizing mice at 10, 30, and 60 min after infusion, we assessed the dynamics of the P40D680 signal within the deep cervical and mandibular lymph nodes of the neck. Deep cervical nodes already had signal at 10 min after the completion of the infusion, as shown in representative images of the left cervical nodes at this time point in Fig.. The signals steadily increased with time, with significantly more tracer found in the nodes at 60 min when compared to 10 min (25,916 ± 10,741 vs. 7723 ± 3333 counts, p < 0.05; one-way ANOVA, Fig. ). Although the mandibular lymph nodes had slightly delayed dynamics, all mice had tracer within the nodes at 30 min after infusion (Fig. ). A significantly increased amount of tracer was found at 60 min compared to 10 min after infusion (28,544 ± 11,942 vs. 2382 ± 1214 counts, p < 0.01; one-way ANOVA, Fig. ). These findings suggest that lymphatic outflow from the CSF is rapid and sustained during the time points examined. Fig. 2 Tracer outflow to draining lymph nodes in the neck. a Re...
We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 tracer near cranial nerve exit routes from the skull at 60 min after intraventricular infusion in Prox1-GFP mice,. Examination of the base of the skull demonstrated accumulation of the tracer around the optic and trigeminal nerves (CN II and V) as they exit the skull (Fig. ). Tracer was also apparent at the cribriform plate that separates the cranial and nasal cavities, indicating that outflow had also occurred along the olfactory nerves (CN I). Removal of the skin around the eye allowed visualization of a dense plexus of tracer-filled lymphatic vessels exiting the orbit that coalesced into one collecting vessel coursing along the anterior facial vein towards the mandibular lymph nodes (Fig. ). There were often vessels that joined into or ran along this collecting vessel that originated from the nasal region. By extraction of the lower jaw and removal of the tongue, esophagus, and trachea, we were able to observe bright signal in the nasal cavity through the palate and tracer emanating fro...
CSF outflow pathways to deep cervical lymph nodes. a Representative image showing lymphatic outflow from the nasal cavity. Lower jaw, tongue, trachea, and esophagus have been removed. pal: palate, phx: pharynx. Inset shows a tracer-filled collecting lymphatic vessel. Scale bar: 2 mm. b Representative image showing outflow from the jugular foramen on the medial side of the tympanic bulla. n: cranial nerves IX, X, and XI, jv: jugular vein, ca: carotid artery. Scale bar: 500 µm. c Representative image showing the perineural outflow along the facial nerve (fn) from the stylomastoid foramen on the lateral side of the tympanic bulla towards a mandibular lymph node (m ln). Scale bar: 1 mm. Images are acquired at t = 60 min after infusion and are representative of n = 10 mice. d Scheme demonstrating the outflow routes in the deep cervical region. Green: lymphatic vessels with black arrows indicating direction of flow, yellow: cranial nerves, red: arteries, blue: veins. Boxes indicates regions of images in a - c and in Fig.
Quantification of lymphatic outflow of CSF has been attempted in many species using approaches such as recovery of radiolabeled tracers through cannulation of collecting lymphatic vessels or detection of signal in draining lymph nodes with imaging approaches,,,. Our above findings have revealed that the multiple routes of lymphatic flow and the anatomical variation between animals indicate that such previous approaches would be limited. Therefore, we aimed to apply a technique recently developed in our lab, which allows quantification of lymphatic transport from an organ by measurement in a peripheral blood vessel of an interstitially injected tracer. This approach assumes that the tracer is lymphatic-specific for the tissue of interest and that the tracer is not retained in the tissue or within draining lymph nodes by phagocytosis. The P40D680 tracer that we have used in the anatomical studies was shown to be highly sensitive for this assay with a direct linear relationship between the amount of intravenously infused tracer and saphenous vein signal, and a detection threshold of ~0.2% of the injected dose in blood (Supplementary Fig. ). We therefore tested whether we...
We infused 2.5 µL of P40D680 into the right lateral ventricle and after 5 min initiated noninvasive imaging of the saphenous vein with NIR stereomicroscopy (Fig. ). The venous signal pattern demonstrated an approximate 25 min delay before signal was seen and a steady increase thereafter (Fig.; Supplementary Movie ). This pattern is indicative of lymph outflow as previously observed in the subcutaneous tissue and the peritoneal cavity. Unlike these tissues, however, outflow was robust from the CSF even under anesthesia, with an estimated 21.6% ± 6.0% ( n = 5 mice) of the infused dose within the bloodstream at 60 min based on the calibration curve shown in Supplementary Fig.. Fig. 5 Dynamics of CSF outflow to systemic blood and mandibular lymph nodes. a Representative GFP and P40D680 images of the saphenous vein region in a Prox1-GFP mouse 60 min after lateral ventricle infusion of P40D680. Prox1-GFP + dermal lymphatic vessels can be observed overlying the saphenous bundle of blood vessels. Scale bars: 500 µm. b Saphenous vein signal enhancement plot of n = 5 mice sh...
We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emission wavelength properties similar to P40D680 were utilized: Evans blue (EB), IRDye680CW, and a 3 kDa dextran conjugated to AlexaFluor680 (3kDa-AF680). Initial experiments were performed to establish comparable doses for intraventricular infusion as well as to test the sensitivity of detection in the saphenous region. It was observed during these studies that although both IRDye680CW and 3kDa-AF680 were not retained within the blood compartment after intravenous injection, they leaked rapidly in sufficient quantities within the skin above the saphenous vein to be detected with high sensitivity (Supplementary Fig. ). In groups of n = 4 mice, we infused 2.5 µL of either 0.6% EB, 200 µM IRDye680CW, or 1.6 mg/ml 3kDa-AF680 into the right lateral ventricle and initiated imaging of the saphenous vein. Surprisingly, we could not detect immediate blood uptake of any tracer, with the patterns exhibiting delays before signal could be detected peripherally (Fig.&#...
The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these disorders was proposed that toxic proteins such as amyloid beta and tau may accumulate in the brain due to reduced clearance. Earlier studies have shown a reduced turnover of CSF and removal of labeled proteins, including amyloid beta, after ventricular-cisternal perfusion in aged rats. Therefore, we tested whether lymphatic outflow from CSF was reduced in aged mice and would be similar to prior observations of reduced peripheral lymphatic transport in aged mice,,. We first tested whether we could detect differences in CSF transport to blood after ventricular infusion of P40D680 in 18-month-old (aged) and 2-month-old (young) C57BL/6J-Tyrc-J albino mice. After 60 min, we observed less P40D680 signal in the saphenous vein of aged mice compared with the young controls (Fig. ). Dynamic imaging (Fig.; Supplementary Movies and ) showed a significant increase in the transit time to blood in aged mice (37.5 ± 8.4 vs. 24.2 ± 2.1 min, p =&#...
For noninvasive imaging of tracer signals in blood, fur above the saphenous vein region was shaved with a razor and depilation cream before the ventricular or cisternal infusion. After removal of the needle from the skull or the cisterna magna, the mice were quickly positioned under a Zeiss StereoLumar.V12 stereomicroscope with AxioVision software (Carl Zeiss, Feldbach, Switzerland) and a Photometrics Evolve 512 camera (Photometrics, Tuscon, AZ) in a supine position on a heating pad to maintain body temperature. The autofluorescence signal on the GFP channel was used to position the saphenous blood vessels at × 25 zoom. Dynamic imaging was initiated 5 min after the completion of the ventricle infusion and 15 min after completion of the cisterna infusion by acquisition of a sequence of images (1 image every 15 s for 55 min) with a Cy5 filter set to monitor the NIR signal of the saphenous vein. Exposure time and camera gain settings were 200 ms and 200, respectively.
Machine-readable layer
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"name": "Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice methods",
"description": "Evidence-backed execution summary for Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice methods from Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice.",
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"text": "After euthanizing mice at 10, 30, and 60 min after infusion, we assessed the dynamics of the P40D680 signal within the deep cervical and mandibular lymph nodes of the neck. Deep cervical nodes already had signal at 10 min after the completion of the infusion, as shown in representative images of the left cervical nodes at this time point in Fig.. The signals steadily increased with time, with significantly more tracer found in the nodes at 60 min when compared to 10 min (25,916 ± 10,741 vs. 7723 ± 3333 counts, p < 0.05; one-way ANOVA, Fig. ). Although the mandibular lymph nodes had slightly delayed dynamics, all mice had tracer within the nodes at 30 min after infusion (Fig. ). A significantly increased amount of tracer was found at 60 min compared to 10 min after infusion (28,5..."
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"name": "Lymphatic routes of CSF from the cranial cavity",
"text": "We next aimed to characterize the lymphatic outflow routes from the murine skull. As perineural outflow pathways have been described in other species, we looked for P40D680 tracer near cranial nerve exit routes from the skull at 60 min after intraventricular infusion in Prox1-GFP mice,. Examination of the base of the skull demonstrated accumulation of the tracer around the optic and trigeminal nerves (CN II and V) as they exit the skull (Fig. ). Tracer was also apparent at the cribriform plate that separates the cranial and nasal cavities, indicating that outflow had also occurred along the olfactory nerves (CN I). Removal of the skin around the eye allowed visualization of a dense plexus of tracer-filled lymphatic vessels exiting the orbit that coalesced into one collecting vessel coursing along the anterior facial vein towards the mandibular lymph nodes (Fig. )...."
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"name": "Lymphatic routes of CSF from the cranial cavity",
"text": "CSF outflow pathways to deep cervical lymph nodes. a Representative image showing lymphatic outflow from the nasal cavity. Lower jaw, tongue, trachea, and esophagus have been removed. pal: palate, phx: pharynx. Inset shows a tracer-filled collecting lymphatic vessel. Scale bar: 2 mm. b Representative image showing outflow from the jugular foramen on the medial side of the tympanic bulla. n: cranial nerves IX, X, and XI, jv: jugular vein, ca: carotid artery. Scale bar: 500 µm. c Representative image showing the perineural outflow along the facial nerve (fn) from the stylomastoid foramen on the lateral side of the tympanic bulla towards a mandibular lymph node (m ln). Scale bar: 1 mm. Images are acquired at t = 60 min after infusion and are representative of n = 10 mice. d Scheme demonstrating the outflow routes in the deep cervical region. Gre..."
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"name": "Lymphatic outflow of a macromolecular tracer from CSF",
"text": "Quantification of lymphatic outflow of CSF has been attempted in many species using approaches such as recovery of radiolabeled tracers through cannulation of collecting lymphatic vessels or detection of signal in draining lymph nodes with imaging approaches,,,. Our above findings have revealed that the multiple routes of lymphatic flow and the anatomical variation between animals indicate that such previous approaches would be limited. Therefore, we aimed to apply a technique recently developed in our lab, which allows quantification of lymphatic transport from an organ by measurement in a peripheral blood vessel of an interstitially injected tracer. This approach assumes that the tracer is lymphatic-specific for the tissue of interest and that the tracer is not retained in the tissue or within draining lymph nodes by phagocytosis. The P40D680 tracer that we have used in the ana..."
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{
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"name": "Lymphatic outflow of a macromolecular tracer from CSF",
"text": "We infused 2.5 µL of P40D680 into the right lateral ventricle and after 5 min initiated noninvasive imaging of the saphenous vein with NIR stereomicroscopy (Fig. ). The venous signal pattern demonstrated an approximate 25 min delay before signal was seen and a steady increase thereafter (Fig.; Supplementary Movie ). This pattern is indicative of lymph outflow as previously observed in the subcutaneous tissue and the peritoneal cavity. Unlike these tissues, however, outflow was robust from the CSF even under anesthesia, with an estimated 21.6% ± 6.0% ( n = 5 mice) of the infused dose within the bloodstream at 60 min based on the calibration curve shown in Supplementary Fig.. Fig. 5 Dynamics of CSF outflow to systemic blood and mandibular lymph nodes. a Representative GFP and P40D680 images of the saph..."
},
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"@type": "HowToStep",
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"name": "CSF outflow of small molecules through the lymphatic system",
"text": "We next determined whether small molecular tracers would exhibit similar outflow patterns compared to P40D680. Three fluorescent small molecular tracers with excitation and emission wavelength properties similar to P40D680 were utilized: Evans blue (EB), IRDye680CW, and a 3 kDa dextran conjugated to AlexaFluor680 (3kDa-AF680). Initial experiments were performed to establish comparable doses for intraventricular infusion as well as to test the sensitivity of detection in the saphenous region. It was observed during these studies that although both IRDye680CW and 3kDa-AF680 were not retained within the blood compartment after intravenous injection, they leaked rapidly in sufficient quantities within the skin above the saphenous vein to be detected with high sensitivity (Supplementary Fig. ). In groups of n = 4 mice, we infused 2.5 µL of either 0.6% EB..."
},
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"@type": "HowToStep",
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"name": "Reduced lymphatic outflow from CSF in aged mice",
"text": "The incidence of Alzheimer's disease and other dementias and many other neurodegenerative diseases increases with age. Recently, a hypothesis for the development of these disorders was proposed that toxic proteins such as amyloid beta and tau may accumulate in the brain due to reduced clearance. Earlier studies have shown a reduced turnover of CSF and removal of labeled proteins, including amyloid beta, after ventricular-cisternal perfusion in aged rats. Therefore, we tested whether lymphatic outflow from CSF was reduced in aged mice and would be similar to prior observations of reduced peripheral lymphatic transport in aged mice,,. We first tested whether we could detect differences in CSF transport to blood after ventricular infusion of P40D680 in 18-month-old (aged) and 2-month-old (young) C57BL/6J-Tyrc-J albino mice. After 60 min, we observed less P40D680 signal i..."
},
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"@type": "HowToStep",
"position": 8,
"name": "Dynamic NIR imaging of CSF outflow",
"text": "For noninvasive imaging of tracer signals in blood, fur above the saphenous vein region was shaved with a razor and depilation cream before the ventricular or cisternal infusion. After removal of the needle from the skull or the cisterna magna, the mice were quickly positioned under a Zeiss StereoLumar.V12 stereomicroscope with AxioVision software (Carl Zeiss, Feldbach, Switzerland) and a Photometrics Evolve 512 camera (Photometrics, Tuscon, AZ) in a supine position on a heating pad to maintain body temperature. The autofluorescence signal on the GFP channel was used to position the saphenous blood vessels at × 25 zoom. Dynamic imaging was initiated 5 min after the completion of the ventricle infusion and 15 min after completion of the cisterna infusion by acquisition of a sequence of images (1 image every 15 s for 55 min) with a Cy5 filter set to monitor..."
}
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"name": "Lymphatic routes of CSF from the cranial cavity"
},
{
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"name": "CSF outflow of small molecules through the lymphatic system"
},
{
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"name": "Infusion of tracers into lateral ventricle or cisterna magna"
},
{
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"name": "Dynamic NIR imaging of CSF outflow"
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"name": "Assessment of transport to blood and lymph nodes"
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"name": "Assessment of collecting lymphatic vessel contractility"
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"name": "Intravenous infusions for tracer dose calibration"
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"name": "In situ analysis of CSF flow and lymphatic outflow"
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"headline": "Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice",
"datePublished": "2017",
"author": [
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"name": "Qiaoli Ma"
},
{
"@type": "Person",
"name": "Benjamin V. Ineichen"
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{
"@type": "Person",
"name": "Michael Detmar"
},
{
"@type": "Person",
"name": "Steven T. Proulx"
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"identifier": "10.1038/s41467-017-01484-6"
}
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