Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry

Yu Zhang1 • Jian Song1 • Xu-Zhong He1 • Jian Xiong2 • Rong Xue1 • Jia-Hao Ge1 • Shi-Yu Lu1 • Die Hu1 • Guo-Xing Zhang1 • Guang-Yin Xu3 • Lin-Hui Wang 1

1 Department of Physiology and Neurobiology, Medical College of Soochow University, Suzhou 215123, China

2 Institute of Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China

3 Institute of Neuroscience, Soochow University, Suzhou 215123, China


Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitative determination of glymphatic activity, and this will be useful in preclinical studies.


Glymphatic system; Cerebrospinal fluid; Fluorescent tracer; Spectrophotofluorometry


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