Michael S Wolf1, Yaming Chen1, Dennis W Simon2, Henry Alexander1, Mark Ross3, Gregory A Gibson3, Miora D Manole4, Hülya Bayır5, Patrick M Kochanek6, Robert S B Clark7. 1. Department of Critical Care Medicine, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, John G. Rangos Research Center - 6th Floor, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA. 2. Department of Critical Care Medicine, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, John G. Rangos Research Center - 6th Floor, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA. 3. Department of Cell Biology, Center for Biologic Imaging, University of Pittsburgh, BST S224, 3500 Terrace Street, Pittsburgh, Pennsylvania, 15261, USA. 4. Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA. 5. Department of Critical Care Medicine, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, John G. Rangos Research Center - 6th Floor, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Department of Environmental and Occupational Health, University of Pittsburgh, 130 De Soto Street, Pittsburgh, Pennsylvania, 15261, USA; Children's Neuroscience Institute, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA. 6. Department of Critical Care Medicine, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, John G. Rangos Research Center - 6th Floor, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Children's Neuroscience Institute, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA. 7. Department of Critical Care Medicine, Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, John G. Rangos Research Center - 6th Floor, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA; Children's Neuroscience Institute, UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, Pennsylvania, 15224, USA. Electronic address: clarkrs@upmc.edu.
Abstract
BACKGROUND: The glymphatic system is a proposed pathway for clearance of proteins and macromolecules from brain, and disrupted glymphatic flux is implicated in neurological disease. We capitalized on colorimetric, fluorescent, and protein-binding properties of Evans blue to evaluate glymphatic flux. NEW METHOD: Twenty-five μL of 1% Evans blue-labeled albumin (EBA) in artificial cerebrospinal fluid (aCSF) was injected into the intracisternal space of anesthetized postnatal day 17 rats. Serum was collected at various time points after injection (n = 37) and EBA was measured spectrophotometrically. In separate rats (n = 3), a cranial window was placed over the parietal cortex and EBA transit was evaluated using in vivo multiphoton microscopy. Separate rats (n = 6) were processed for immunohistochemistry to examine localization of EBA. In some rats, intracranial pressure (ICP) was increased via intracisternal injection of aCSF. RESULTS: EBA was detected in serum as early as 30 min, was maximal at 4 h, and was undetectable at 72 h after intracisternal injection. Using intra-vital microscopy and immunohistochemistry EBA could be tracked from CSF to perivascular locations. Consistent with removal via glymphatic flux, increasing ICP to 40 mmHg accelerated transit of EBA from CSF to blood. COMPARISON WITH EXISTING METHODS: Transit of EBA from CSF to serum could be quantified spectrophotometrically without radioactive labeling. Glymphatic flux could also be qualitatively evaluated using EBA fluorescence. CONCLUSION: We present a novel technique for simultaneous quantitative and qualitative evaluation of glymphatic flux in rats.
BACKGROUND: The glymphatic system is a proposed pathway for clearance of proteins and macromolecules from brain, and disrupted glymphatic flux is implicated in neurological disease. We capitalized on colorimetric, fluorescent, and protein-binding properties of Evans blue to evaluate glymphatic flux. NEW METHOD: Twenty-five μL of 1% Evans blue-labeled albumin (EBA) in artificial cerebrospinal fluid (aCSF) was injected into the intracisternal space of anesthetized postnatal day 17 rats. Serum was collected at various time points after injection (n = 37) and EBA was measured spectrophotometrically. In separate rats (n = 3), a cranial window was placed over the parietal cortex and EBA transit was evaluated using in vivo multiphoton microscopy. Separate rats (n = 6) were processed for immunohistochemistry to examine localization of EBA. In some rats, intracranial pressure (ICP) was increased via intracisternal injection of aCSF. RESULTS:EBA was detected in serum as early as 30 min, was maximal at 4 h, and was undetectable at 72 h after intracisternal injection. Using intra-vital microscopy and immunohistochemistry EBA could be tracked from CSF to perivascular locations. Consistent with removal via glymphatic flux, increasing ICP to 40 mmHg accelerated transit of EBA from CSF to blood. COMPARISON WITH EXISTING METHODS: Transit of EBA from CSF to serum could be quantified spectrophotometrically without radioactive labeling. Glymphatic flux could also be qualitatively evaluated using EBA fluorescence. CONCLUSION: We present a novel technique for simultaneous quantitative and qualitative evaluation of glymphatic flux in rats.
Authors: M J Whalen; T M Carlos; P M Kochanek; R S Clark; S Heineman; J K Schiding; D Franicola; F Memarzadeh; W Lo; D W Marion; S T Dekosky Journal: J Neurotrauma Date: 1999-07 Impact factor: 5.269
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