Circadian control of brain glymphatic and lymphatic fluid flow methods
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Results
Glymphatic influx does not reflect function of the whole glymphatic system, therefore we measured clearance of Evans blue (EB), a small molecule (960 Da) that freely diffuses through the brain and binds tightly to albumin in the blood, from the brain to the femoral vein in anesthetized animals during the day ( n = 9 mice) and night ( n = 6 mice). We chose this method because although we cannot measure where in the vasculature EB enters and binds to albumin, any fluorescence detected in the femoral vein had to be cleared from the brain parenchyma. Thus, this approach enabled us to test the hypothesis that net clearance of EB from the brain is under diurnal control. Mice were implanted with a microdialysis cannula into the striatum 24-36 h prior to experiments. At the time of experiment, the microdialysis probe was inserted in the cannula, the...
Methods
Male and female C57BL/6 mice (aged 3-5 months, weight between 25 g and 30 g) were acquired from Charles River Laboratories (Wilmington, MA) in equal numbers for each experimental group to control for any potential sex differences. Male and female AQP4 KO mice were bred in the University of Rochester vivarium, and backcrossed to C57BL/6 mice for 20+ generations before use. A minimum of five mice were used in each group, with exact animal numbers stated in the results and figure legends. Mice were group-housed either in a 12:12 light/dark cycle or under constant light with ad libitum access to food and water. All experiments were approved by the University of Rochester Medical Center Committee on Animal Resources. All of the University of Rochester's animal holding rooms are maintained within temperature (18-26 degrees Celsius) and humidity ranges (30-...
Methods
For constant light experiments, animals were housed two per cage to reduce risk of hypothermia. Activity was monitored continuously in 5 min bins via the Comprehensive Lab Animal Monitoring System (Columbus Instruments). Circadian behavioral analysis was completed with ActogramJ. Free-running period of each cage was determined using at least 10 days of activity and a χ 2 periodogram, which was used to confirm behavioral rhythmicity and estimate experimental times in conjunction with activity onset of the cage.
Intracisternal CSF tracer infusion
Fluorescent CSF tracer (bovine serum albumin, Alexa Fluor TM 647 conjugate; 66 kDa; Invitrogen, Life Technologies, Eugene, OR) was formulated in artificial CSF at a concentration of 0.5% weight by volume. Anesthetized mice were fixed in a stereotaxic frame, the CM surgically exposed, and a 30 gauge needle connected to PE10 tubing filled with the tracer was inserted into the CM. Ten microliters of CSF tracer was infused at a rate of 2 µl min -1 for 5 min with a syringe pump (Harvard Apparatus). For the awake animal cohort: mice were anesthetized with 1-2% isoflurane, and 0.25% bupivicane was applied topically to the surgery site at the beginning of and after recovery from the procedure. Mice were allowed to resurface from anesthesia before pump start. Total volume of CSF tracer was increased to 12 µL to compensate for decreased glymp...
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Evidence quotes (4)
Glymphatic influx does not reflect function of the whole glymphatic system, therefore we measured clearance of Evans blue (EB), a small molecule (960 Da) that freely diffuses through the brain and binds tightly to albumin in the blood, from the brain to the femoral vein in anesthetized animals during the day ( n = 9 mice) and night ( n = 6 mice). We chose this method because although we cannot measure where in the vasculature EB enters and binds to albumin, any fluorescence detected in the femoral vein had to be cleared from the brain parenchyma. Thus, this approach enabled us to test the hypothesis that net clearance of EB from the brain is under diurnal control. Mice were implanted with a microdialysis cannula into the striatum 24-36 h prior to experiments. At the time of experiment, the microdialysis probe was inserted in the cannula, the femoral vein exposed, and placed under the macroscope (Fig. ). Imaging began at the beginning of infusion of EB (4% in ACSF, 1 µL, 0.2 µL min -1 ). EB was significantly ( t test: t (13) = 2.605, p = 0.0218) higher in the femoral vein...
Male and female C57BL/6 mice (aged 3-5 months, weight between 25 g and 30 g) were acquired from Charles River Laboratories (Wilmington, MA) in equal numbers for each experimental group to control for any potential sex differences. Male and female AQP4 KO mice were bred in the University of Rochester vivarium, and backcrossed to C57BL/6 mice for 20+ generations before use. A minimum of five mice were used in each group, with exact animal numbers stated in the results and figure legends. Mice were group-housed either in a 12:12 light/dark cycle or under constant light with ad libitum access to food and water. All experiments were approved by the University of Rochester Medical Center Committee on Animal Resources. All of the University of Rochester's animal holding rooms are maintained within temperature (18-26 degrees Celsius) and humidity ranges (30-70%) described in the ILAR Guide for the Care and Use of Laboratory Animals (1996). All efforts were made to keep animal usage to a minimum.
For constant light experiments, animals were housed two per cage to reduce risk of hypothermia. Activity was monitored continuously in 5 min bins via the Comprehensive Lab Animal Monitoring System (Columbus Instruments). Circadian behavioral analysis was completed with ActogramJ. Free-running period of each cage was determined using at least 10 days of activity and a χ 2 periodogram, which was used to confirm behavioral rhythmicity and estimate experimental times in conjunction with activity onset of the cage.
Fluorescent CSF tracer (bovine serum albumin, Alexa Fluor TM 647 conjugate; 66 kDa; Invitrogen, Life Technologies, Eugene, OR) was formulated in artificial CSF at a concentration of 0.5% weight by volume. Anesthetized mice were fixed in a stereotaxic frame, the CM surgically exposed, and a 30 gauge needle connected to PE10 tubing filled with the tracer was inserted into the CM. Ten microliters of CSF tracer was infused at a rate of 2 µl min -1 for 5 min with a syringe pump (Harvard Apparatus). For the awake animal cohort: mice were anesthetized with 1-2% isoflurane, and 0.25% bupivicane was applied topically to the surgery site at the beginning of and after recovery from the procedure. Mice were allowed to resurface from anesthesia before pump start. Total volume of CSF tracer was increased to 12 µL to compensate for decreased glymphatic function, pump speed remained the same (2 µl min -1 ).
Machine-readable layer
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