Quantitative and qualitative assessment of glymphatic flux using Evans blue albumin

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.

Pituitary Adenylate Cyclase-Activating Polypeptide Attenuates Brain Edema by Protecting Blood–Brain Barrier and Glymphatic System After Subarachnoid Hemorrhage in Rats

“…Cisterna magna injection of EB dye was conducted to observe the movement of CSF after SAH as previously described [36]. Anesthetized rats were placed in the stereotaxic frame, and the atlantooccipital membrane was exposed by a midline incision.…”

Impaired neural differentiation and glymphatic CSF flow in the Ccdc39 rat model of neonatal hydrocephalus: genetic interaction with L1cam

“…Evans Blue is a low molecular weight tracing dye with a high affinity for serum albumin that accumulates in perivascular spaces, such as the Virchow–Robin spaces of the glymphatic system, after intracerebral injection (Maloveska et al, 2018; Stoelinga and van Munster, 1967). It has been used as a marker for blood–brain barrier integrity, meningeal lymphatic vasculature and glymphatic flux because of its colorimetric, fluorescent and protein-binding qualities (Maloveska et al, 2018; Stoelinga and van Munster, 1967; Wolf et al, 2019). Likewise, various groups have used fluorescently labeled protein tracers injected into the cisterna magna to show flux of labeled proteins in the CSF through the glymphatic system (Iliff et al, 2012; Ma et al, 2017).…”

Dysfunctional glymphatic system with disrupted aquaporin-4 expression pattern on astrocytes causes bacterial product accumulation in the CSF during pneumococcal meningitis

“…The previous anesthetized animal that received the bacterial suspension or aCSF was placed in the stereotaxic on a heating pad, and the head was fixed on the apparatus with the rat’s nose slightly pointed downwards. The cannula needle was inserted (approximately 1 to 2 mm) into the center of the cisterna magna, and it started the injection of EBA using the microinjection syringe pump at a rate of 1 μL per minute for 25 min, resulting in a total volume injected of 25 μL of 1% EBA solution [58].…”