Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma
Alex J Smith, Xiaoming Yao, James A Dix, Byung-Ju Jin, Alan S Verkman, Alex J Smith, Xiaoming Yao, James A Dix, Byung-Ju Jin, Alan S Verkman
Abstract
Transport of solutes through brain involves diffusion and convection. The importance of convective flow in the subarachnoid and paravascular spaces has long been recognized; a recently proposed 'glymphatic' clearance mechanism additionally suggests that aquaporin-4 (AQP4) water channels facilitate convective transport through brain parenchyma. Here, the major experimental underpinnings of the glymphatic mechanism were re-examined by measurements of solute movement in mouse brain following intracisternal or intraparenchymal solute injection. We found that: (i) transport of fluorescent dextrans in brain parenchyma depended on dextran size in a manner consistent with diffusive rather than convective transport; (ii) transport of dextrans in the parenchymal extracellular space, measured by 2-photon fluorescence recovery after photobleaching, was not affected just after cardiorespiratory arrest; and (iii) Aqp4 gene deletion did not impair transport of fluorescent solutes from sub-arachnoid space to brain in mice or rats. Our results do not support the proposed glymphatic mechanism of convective solute transport in brain parenchyma.
Keywords: CSF; aquaporin 4; convection; diffusion; extracellular space; glymphatic hypothesis; mouse; neuroscience; rat.
Conflict of interest statement
No competing interests declared.
Figures
References
- Abbott NJ. Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology. Neurochemistry International. 2004;45:545–552. doi: 10.1016/j.neuint.2003.11.006.
- Asgari M, de Zélicourt D, Kurtcuoglu V. How astrocyte networks may contribute to cerebral metabolite clearance. Scientific Reports. 2015;5:15024. doi: 10.1038/srep15024.
- Asgari M, de Zélicourt D, Kurtcuoglu V. Glymphatic solute transport does not require bulk flow. Scientific Reports. 2016;6:38635. doi: 10.1038/srep38635.
- Bakker EN, Bacskai BJ, Arbel-Ornath M, Aldea R, Bedussi B, Morris AW, Weller RO, Carare RO. Lymphatic clearance of the brain: perivascular, paravascular and significance for neurodegenerative diseases. Cellular and Molecular Neurobiology. 2016;36:181–194. doi: 10.1007/s10571-015-0273-8.
- Bedussi B, van Lier MG, Bartstra JW, de Vos J, Siebes M, VanBavel E, Bakker EN. Clearance from the mouse brain by convection of interstitial fluid towards the ventricular system. Fluids and Barriers of the CNS. 2015;12:23. doi: 10.1186/s12987-015-0019-5.
- Bedussi B, van der Wel NN, de Vos J, van Veen H, Siebes M, VanBavel E, Bakker ENTP. Paravascular channels, cisterns, and the subarachnoid space in the rat brain: A single compartment with preferential pathways. Journal of Cerebral Blood Flow & Metabolism. 2017;37:1374–1385. doi: 10.1177/0271678X16655550.
- Binder DK, Papadopoulos MC, Haggie PM, Verkman AS. In vivo measurement of brain extracellular space diffusion by cortical surface photobleaching. Journal of Neuroscience. 2004;24:8049–8056. doi: 10.1523/JNEUROSCI.2294-04.2004.
- Dechadilok P, Deen WM. Electrostatic and electrokinetic effects on hindered convection in pores. Journal of Colloid and Interface Science. 2009;338:135–144. doi: 10.1016/j.jcis.2009.06.018.
- Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behavior Research Methods. 2009;41:1149–1160. doi: 10.3758/BRM.41.4.1149.
- Foley CP, Nishimura N, Neeves KB, Schaffer CB, Olbricht WL. Real-time imaging of perivascular transport of nanoparticles during convection-enhanced delivery in the rat cortex. Annals of Biomedical Engineering. 2012;40:292–303. doi: 10.1007/s10439-011-0440-0.
- Hadjiev NA, Amsden BG. An assessment of the ability of the obstruction-scaling model to estimate solute diffusion coefficients in hydrogels. Journal of Controlled Release. 2015;199:10–16. doi: 10.1016/j.jconrel.2014.12.010.
- Hladky SB, Barrand MA. Mechanisms of fluid movement into, through and out of the brain: evaluation of the evidence. Fluids and Barriers of the CNS. 2014;11:26. doi: 10.1186/2045-8118-11-26.
- Holtmaat A, Bonhoeffer T, Chow DK, Chuckowree J, De Paola V, Hofer SB, Hübener M, Keck T, Knott G, Lee WC, Mostany R, Mrsic-Flogel TD, Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg JT, Wilbrecht L. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nature Protocols. 2009;4:1128–1144. doi: 10.1038/nprot.2009.89.
- Hong S, Ostaszewski BL, Yang T, O'Malley TT, Jin M, Yanagisawa K, Li S, Bartels T, Selkoe DJ. Soluble Aβ oligomers are rapidly sequestered from brain ISF in vivo and bind GM1 ganglioside on cellular membranes. Neuron. 2014;82:308–319. doi: 10.1016/j.neuron.2014.02.027.
- Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Science Translational Medicine. 2012;4:147ra111. doi: 10.1126/scitranslmed.3003748.
- Iliff JJ, Wang M, Zeppenfeld DM, Venkataraman A, Plog BA, Liao Y, Deane R, Nedergaard M. Cerebral arterial pulsation drives paravascular CSF-interstitial fluid exchange in the murine brain. Journal of Neuroscience. 2013;33:18190–18199. doi: 10.1523/JNEUROSCI.1592-13.2013.
- Iliff JJ, Chen MJ, Plog BA, Zeppenfeld DM, Soltero M, Yang L, Singh I, Deane R, Nedergaard M. Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury. The Journal of Neuroscience. 2014;34:16180–16193. doi: 10.1523/JNEUROSCI.3020-14.2014.
- Jessen NA, Munk AS, Lundgaard I, Nedergaard M. The glymphatic system: a beginner's guide. Neurochemical Research. 2015;40:2583–2599. doi: 10.1007/s11064-015-1581-6.
- Jin BJ, Smith AJ, Verkman AS. Spatial model of convective solute transport in brain extracellular space does not support a "glymphatic" mechanism. The Journal of General Physiology. 2016;148:489–501. doi: 10.1085/jgp.201611684.
- Korogod N, Petersen CCH, Knott GW. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation. eLife. 2015;4:e05793. doi: 10.7554/eLife.05793.
- Kress BT, Iliff JJ, Xia M, Wang M, Wei HS, Zeppenfeld D, Xie L, Kang H, Xu Q, Liew JA, Plog BA, Ding F, Deane R, Nedergaard M. Impairment of paravascular clearance pathways in the aging brain. Annals of Neurology. 2014;76:845–861. doi: 10.1002/ana.24271.
- Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. Journal of Clinical Investigation. 1997;100:957–962. doi: 10.1172/JCI231.
- Mazza D, Braeckmans K, Cella F, Testa I, Vercauteren D, Demeester J, De Smedt SS, Diaspro A. A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy. Biophysical Journal. 2008;95:3457–3469. doi: 10.1529/biophysj.108.133637.
- McBain CJ, Traynelis SF, Dingledine R. Regional variation of extracellular space in the hippocampus. Science. 1990;249:674–677. doi: 10.1126/science.2382142.
- Morris AW, Sharp MM, Albargothy NJ, Fernandes R, Hawkes CA, Verma A, Weller RO, Carare RO. Vascular basement membranes as pathways for the passage of fluid into and out of the brain. Acta Neuropathologica. 2016;131:725–736. doi: 10.1007/s00401-016-1555-z.
- Nicholson C, Tao L. Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging. Biophysical Journal. 1993;65:2277–2290. doi: 10.1016/S0006-3495(93)81324-9.
- Papadopoulos MC, Kim JK, Verkman AS. Extracellular space diffusion in central nervous system: anisotropic diffusion measured by elliptical surface photobleaching. Biophysical Journal. 2005;89:3660–3668. doi: 10.1529/biophysj.105.068114.
- Rennels ML, Blaumanis OR, Grady PA. Rapid solute transport throughout the brain via paravascular fluid pathways. Advances in Neurology. 1990;52:431–439.
- Risher WC, Andrew RD, Kirov SA. Real-time passive volume responses of astrocytes to acute osmotic and ischemic stress in cortical slices and in vivo revealed by two-photon microscopy. Glia. 2009;57:207–221. doi: 10.1002/glia.20747.
- Rosenberg GA, Kyner WT, Estrada E. Bulk flow of brain interstitial fluid under normal and hyperosmolar conditions. The American Journal of Physiology. 1980;238:F42–49.
- Sandrin D, Wagner D, Sitta CE, Thoma R, Felekyan S, Hermes HE, Janiak C, de Sousa Amadeu N, Kühnemuth R, Löwen H, Egelhaaf SU, Seidel CA. Diffusion of macromolecules in a polymer hydrogel: from microscopic to macroscopic scales. Phys. Chem. Chem. Phys. 2016;18:12860–12876. doi: 10.1039/C5CP07781H.
- Smith AJ, Jin BJ, Verkman AS. Muddying the water in brain edema? Trends in Neurosciences. 2015;38:331–332. doi: 10.1016/j.tins.2015.04.006.
- Smith AJ, Yao X, Dix JA, Jin BJ, Verkman AS. Convection-diffusion-analysis. [3bc0f60];Github. 2017
- Spector R, Robert Snodgrass S, Johanson CE. A balanced view of the cerebrospinal fluid composition and functions: focus on adult humans. Experimental Neurology. 2015;273:57–68. doi: 10.1016/j.expneurol.2015.07.027.
- Steiner E, Enzmann GU, Lin S, Ghavampour S, Hannocks MJ, Zuber B, Rüegg MA, Sorokin L, Engelhardt B. Loss of astrocyte polarization upon transient focal brain ischemia as a possible mechanism to counteract early edema formation. Glia. 2012;60:1646–1659. doi: 10.1002/glia.22383.
- Sullivan KD, Sipprell WH, Brown EB, Brown EB. Improved model of fluorescence recovery expands the application of multiphoton fluorescence recovery after photobleaching in vivo. Biophysical Journal. 2009;96:5082–5094. doi: 10.1016/j.bpj.2009.04.020.
- Syková E, Svoboda J, Polák J, Chvátal A. Extracellular volume fraction and diffusion characteristics during progressive ischemia and terminal anoxia in the spinal cord of the rat. Journal of Cerebral Blood Flow & Metabolism. 1994;14:301–311. doi: 10.1038/jcbfm.1994.37.
- Syková E, Nicholson C. Diffusion in brain extracellular space. Physiological Reviews. 2008;88:1277–1340. doi: 10.1152/physrev.00027.2007.
- Tao L, Nicholson C. Diffusion of albumins in rat cortical slices and relevance to volume transmission. Neuroscience. 1996;75:839–847. doi: 10.1016/0306-4522(96)00303-X.
- Tarasoff-Conway JM, Carare RO, Osorio RS, Glodzik L, Butler T, Fieremans E, Axel L, Rusinek H, Nicholson C, Zlokovic BV, Frangione B, Blennow K, Ménard J, Zetterberg H, Wisniewski T, de Leon MJ. Clearance systems in the brain-implications for Alzheimer disease. Nature Reviews Neurology. 2015;11:457–470. doi: 10.1038/nrneurol.2015.119.
- Thrane AS, Rangroo Thrane V, Nedergaard M. Drowning stars: reassessing the role of astrocytes in brain edema. Trends in Neurosciences. 2014;37:620–628. doi: 10.1016/j.tins.2014.08.010.
- Verkman AS. Diffusion in the extracellular space in brain and tumors. Physical Biology. 2013;10:045003. doi: 10.1088/1478-3975/10/4/045003.
- Wilcock DM, Vitek MP, Colton CA. Vascular amyloid alters astrocytic water and potassium channels in mouse models and humans with Alzheimer's disease. Neuroscience. 2009;159:1055–1069. doi: 10.1016/j.neuroscience.2009.01.023.
- Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, O'Donnell J, Christensen DJ, Nicholson C, Iliff JJ, Takano T, Deane R, Nedergaard M. Sleep drives metabolite clearance from the adult brain. Science. 2013;342:373–377. doi: 10.1126/science.1241224.
- Xu Z, Xiao N, Chen Y, Huang H, Marshall C, Gao J, Cai Z, Wu T, Hu G, Xiao M. Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits. Molecular Neurodegeneration. 2015;10:58. doi: 10.1186/s13024-015-0056-1.
- Yao X, Hrabetová S, Nicholson C, Manley GT. Aquaporin-4-deficient mice have increased extracellular space without tortuosity change. Journal of Neuroscience. 2008;28:5460–5464. doi: 10.1523/JNEUROSCI.0257-08.2008.
- Zhang H, Verkman AS. Microfiberoptic measurement of extracellular space volume in brain and tumor slices based on fluorescent dye partitioning. Biophysical Journal. 2010;99:1284–1291. doi: 10.1016/j.bpj.2010.06.023.
- Zoremba N, Homola A, Slais K, Vorísek I, Rossaint R, Lehmenkühler A, Syková E. Extracellular diffusion parameters in the rat somatosensory cortex during recovery from transient global ischemia/hypoxia. Journal of Cerebral Blood Flow & Metabolism. 2008;28:1665–1673. doi: 10.1038/jcbfm.2008.58.
Source: PubMed