Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry

Abstract

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.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31871167 and 81920108016), China Postdoctoral Science Foundation (2016M601882), Suzhou Science and Technology Research Project (SYS201669 and SYS201709), Postdoctoral Science Foundation of Jiangsu Province, China (1601083C), and Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Correspondence to Guang-Yin Xu or Lin-Hui Wang.

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Zhang, Y., Song, J., He, XZ. et al. Quantitative Determination of Glymphatic Flow Using Spectrophotofluorometry. Neurosci. Bull. 36, 1524–1537 (2020). https://doi.org/10.1007/s12264-020-00548-w

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Keywords

  • Glymphatic system
  • Cerebrospinal fluid
  • Fluorescent tracer
  • Spectrophotofluorometry