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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med 2012, 4: 147ra111.
Rasmussen MK, Mestre H, Nedergaard M. The glymphatic pathway in neurological disorders. Lancet Neurol 2018, 17: 1016–1024.
Jessen NA, Munk AS, Lundgaard I, Nedergaard M. The glymphatic system: a beginner’s guide. Neurochem Res 2015, 40: 2583–2599.
Iliff JJ, Chen MJ, Plog BA, Zeppenfeld DM, Soltero M, Yang L, et al. Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury. J Neurosci 2014, 34: 16180–16193.
Lundgaard I, Lu ML, Yang E, Peng W, Mestre H, Hitomi E, et al. Glymphatic clearance controls state-dependent changes in brain lactate concentration. J Cereb Blood Flow Metab 2017, 37: 2112–2124.
Achariyar TM, Li B, Peng W, Verghese PB, Shi Y, McConnell E, et al. Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation. Mol Neurodegener 2016, 11: 74.
Lundgaard I, Li B, Xie L, Kang H, Sanggaard S, Haswell JD, et al. Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism. Nat Commun 2015, 6: 6807.
Rangroo Thrane V, Thrane AS, Plog BA, Thiyagarajan M, Iliff JJ, Deane R, et al. Paravascular microcirculation facilitates rapid lipid transport and astrocyte signaling in the brain. Sci Rep 2013, 3: 2582.
Benveniste H, Lee H, Volkow ND. The glymphatic pathway: waste removal from the CNS via cerebrospinal fluid transport. Neuroscientist 2017, 23: 454–465.
Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, et al. Sleep drives metabolite clearance from the adult brain. Science 2013, 342: 373–377.
Lee H, Xie L, Yu M, Kang H, Feng T, Deane R, et al. The effect of body posture on brain glymphatic transport. J Neurosci 2015, 35: 11034–11044.
Hablitz LM, Vinitsky HS, Sun Q, Staeger FF, Sigurdsson B, Mortensen KN, et al. Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia. Sci Adv 2019, 5: eaav5447.
Kress BT, Iliff JJ, Xia MS, Wang MH, Wei HLS, Zeppenfeld D, et al. Impairment of paravascular clearance pathways in the aging brain. Ann Neurol 2014, 76: 845–861.
Peng WG, Achariyar TM, Li BM, Liao YH, Mestre H, Hitomi E, et al. Suppression of glymphatic fluid transport in a mouse model of Alzheimer’s disease. Neurobiol Disease 2016, 93: 215–225.
Gaberel T, Gakuba C, Goulay R, Martinez De Lizarrondo S, Hanouz JL, Emery E, et al. Impaired glymphatic perfusion after strokes revealed by contrast-enhanced MRI: a new target for fibrinolysis? Stroke 2014, 45: 3092–3096.
Mestre H, Tithof J, Du T, Song W, Peng W, Sweeney AM, et al. Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nat Commun 2018, 9: 4878.
Nygaard Mortensen K, Sanggaard S, Mestre H, Lee H, Kostrikov S, Xavier ALR, et al. Impaired glymphatic transport in spontaneously hypertensive rats. J Neurosci 2019, 39: 6365–6377.
Yang L, Kress BT, Weber HJ, Thiyagarajan M, Wang B, Deane R, et al. Evaluating glymphatic pathway function utilizing clinically relevant intrathecal infusion of CSF tracer. J Transl Med 2013, 11: 107.
Wei F, Song J, Zhang C, Lin J, Xue R, Shan LD, et al. Chronic stress impairs the aquaporin-4-mediated glymphatic transport through glucocorticoid signaling. Psychopharmacology (Berl) 2019.
Wei F, Zhang C, Xue R, Shan L, Gong S, Wang G, et al. The pathway of subarachnoid CSF moving into the spinal parenchyma and the role of astrocytic aquaporin-4 in this process. Life Sci 2017, 182: 29–40.
Zhang C, Lin J, Wei F, Song J, Chen W, Shan L, et al. Characterizing the glymphatic influx by utilizing intracisternal infusion of fluorescently conjugated cadaverine. Life Sci 2018, 201: 150–160.
Smith AJ, Yao X, Dix JA, Jin BJ, Verkman AS. Test of the ‘glymphatic’ hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma. Elife 2017, 6.
Schain AJ, Melo-Carrillo A, Strassman AM, Burstein R. Cortical spreading depression closes paravascular space and impairs glymphatic flow: implications for migraine headache. J Neurosci 2017, 37: 2904–2915.
Plog BA, Mestre H, Olveda GE, Sweeney AM, Kenney HM, Cove A, et al. Transcranial optical imaging reveals a pathway for optimizing the delivery of immunotherapeutics to the brain. JCI Insight 2018, 3.
Pizzo ME, Wolak DJ, Kumar NN, Brunette E, Brunnquell CL, Hannocks MJ, et al. Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery. J Physiol 2018, 596: 445–475.
Saria A, Lundberg JM. Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues. J Neurosci Methods 1983, 8: 41–49.
Wolf MS, Chen Y, Simon DW, Alexander H, Ross M, Gibson GA, et al. Quantitative and qualitative assessment of glymphatic flux using Evans blue albumin. J Neurosci Methods 2019, 311: 436–441.
Dorshow RB, Hall-Moore C, Shaikh N, Talcott MR, Faubion WA, Rogers TE, et al. Measurement of gut permeability using fluorescent tracer agent technology. Sci Rep 2017, 7: 10888.
Wei F, Song J, Zhang C, Lin J, Xue R, Shan LD, et al. Chronic stress impairs the aquaporin-4-mediated glymphatic transport through glucocorticoid signaling. Psychopharmacology (Berl) 2019, 236: 1367–1384.
Gao Y, Wang Z, Zhu Y, Zhu Q, Yang Y, Jin Y, et al. NOP2/Sun RNA methyltransferase 2 promotes tumor progression via its interacting partner RPL6 in gallbladder carcinoma. Cancer Sci 2019, 110: 3510–3519.
Dos Santos TG, Pereira MSL, Oliveira DL. Rat cerebrospinal fluid treatment method through cisterna cerebellomedullaris injection. Neurosci Bull 2018, 34: 827–832.
Liu T, Li Z, He J, Yang N, Han D, Li Y, et al. Regional metabolic patterns of abnormal postoperative behavioral performance in aged mice assessed by (1)H-NMR dynamic mapping method. Neurosci Bull 2020, 36: 25–38.
Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, et al. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. J Clin Invest 2013, 123: 1299–1309.
Ringstad G, Vatnehol SAS, Eide PK. Glymphatic MRI in idiopathic normal pressure hydrocephalus. Brain 2017, 140: 2691–2705.
de Leon MJ, Li Y, Okamura N, Tsui WH, Saint-Louis LA, Glodzik L, et al. Cerebrospinal fluid clearance in Alzheimer disease measured with dynamic PET. J Nucl Med 2017, 58: 1471–1476.
Lundgaard I, Wang W, Eberhardt A, Vinitsky HS, Reeves BC, Peng S, et al. Beneficial effects of low alcohol exposure, but adverse effects of high alcohol intake on glymphatic function. Sci Rep 2018, 8: 2246.
von Holstein-Rathlou S, Petersen NC, Nedergaard M. Voluntary running enhances glymphatic influx in awake behaving, young mice. Neurosci Lett 2018, 662: 253–258.
Munk AS, Wang W, Bechet NB, Eltanahy AM, Cheng AX, Sigurdsson B, et al. PDGF-B is required for development of the glymphatic system. Cell Rep 2019, 26: 2955-2969 e2953.
Xia M, Yang L, Sun G, Qi S, Li B. Mechanism of depression as a risk factor in the development of Alzheimer’s disease: the function of AQP4 and the glymphatic system. Psychopharmacology (Berl) 2017, 234: 365–379.
Dai JK, Wang SX, Shan D, Niu HC, Lei H. Super-resolution track-density imaging reveals fine anatomical features in tree shrew primary visual cortex and hippocampus. Neurosci Bull 2018, 34: 438–448.
Mestre H, Hablitz LM, Xavier AL, Feng W, Zou W, Pu T, et al. Aquaporin-4-dependent glymphatic solute transport in the rodent brain. Elife 2018, 7.
Deane R, Wu Z, Sagare A, Davis J, Du Yan S, Hamm K, et al. LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms. Neuron 2004, 43: 333–344.
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.
About this article
Cite this article
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
- Glymphatic system
- Cerebrospinal fluid
- Fluorescent tracer