Determining the relative importance of convection versus diffusion in transporting solutes through brain extracellular space is a long-standing problem in neurobiology of particular importance for clearance of toxic protein aggregates (Hladky and Barrand, 2014; Bakker et al., 2016; Abbott, 2004). Some amount of convective flow in the paravascular spaces is generally accepted, although the direction of this flow, its magnitude, and the mechanisms that regulate it remain less clear (Hladky and Barrand, 2014). Whereas most previous authors have concluded that solute movement in brain parenchyma is adequately described by simple diffusion, Nedergaard and colleagues proposed that convective flow in the para-arterial spaces continues into the parenchyma resulting in directional clearance of solutes into the paravenous spaces, by a mechanism requiring AQP4 expression in astrocyte endfeet. Here, we independently tested several major predictions of the glymphatic hypothesis and find that: (i) Aqp4 deletion does not impair transfer of solutes from CSF into the parenchyma; (ii) movement of fluorescent solutes of different sizes through brain parenchyma is consistent with their diffusion coefficients; and (iii) local movement of solutes in the parenchyma is not impaired just after cardiorespiratory arrest. These results do not support glymphatic, convective solute transport in brain parenchyma.

The experiments reported here add to recent work describing the fate of tracer molecules injected into the brain (Bedussi et al., 2015; Morris et al., 2016; Bedussi et al., 2017). We found size-independent distribution of cisternally injected dextrans into the paravascular spaces, but strongly size-dependent dextran penetration into the parenchyma. These results support convective solute transport in paravascular pathways, but provide evidence against a substantial role for convection in brain parenchyma. An interesting observation was that the smallest (10 kDa) dextran was seen in both the paravascular space between astrocytes and vascular smooth muscle and in the ‘perivascular’ space between the smooth muscle and endothelium, according to the terminology of Bakker et al. (Bakker et al., 2016). It remains unclear if accumulation of the small dextran in the perivascular space is due to size-dependent permeability of the living vessel or, alternatively, the early stages of clearance of the low molecular weight dextran along a separate pathway in the peri-arteriolar smooth muscle ring as recently suggested by Morris et al. (Morris et al., 2016). Direct injection of dextrans into the striatum resulted in their symmetrical, size-dependent movement away from the injection site in a manner consistent with pure diffusion. As with cisternal injections, size-independent accumulation of tracers was observed in the paravascular spaces, consistent with previous measurements of the transport of large solutes during convection-enhanced delivery (Foley et al., 2012). The relative homogeneity of the striatum results in approximately isotropic diffusion; however, this is unlikely to be the case in more organized regions of the brain where structural barriers, or regional variation in extracellular space fraction (McBain et al., 1990), will likely produce anisotropic diffusion. In white matter tracts, both diffusional anisotropy (Papadopoulos et al., 2005) and slow convection (Rosenberg et al., 1980) have been demonstrated.

Similar observations were made, with dye visible in both the parenchyma and paravascular areas, when a larger volume of dextran was injected into the cortex and visualized by 2-photon imaging through a cranial window (Figure 3A). Recent electron microscopy-level observations of astrocyte fine structure in vitrified samples, where tissue shrinkage during fixation is minimal, demonstrate that the paravascular space may be larger than previously appreciated and that endfoot coverage of vessels is incomplete (Korogod et al., 2015). The apparent enrichment of dye around vessels is therefore consistent with a diffusional equilibrium between a fluid-filled paravascular space and a parenchyma with ECS volume fraction of ~0.2. We did not observe directional solute transport in the parenchyma in photobleaching experiments, where recovery of fluorescence in bleached disks was consistently isotropic. Reduction in the rate of solute transfer from the paravascular space to the parenchyma following unilateral internal carotid artery ligation led Iliff et al. (Iliff et al., 2013) to conclude that cerebral arterial pulsatility is a key driver of paravascular solute influx into and through brain parenchyma. Although the role of arterial pulsation in moving CSF in the subarachnoid and paravascular spaces is well-established (Rennels et al., 1990), it had not been previously been suggested that arterial pulsation could drive fluid flow through the interstitial space. We directly assessed the contribution of arterial pulsatility to solute movement in the interstitial space by photobleaching of 150 kDa FITC-dextran and found that solute transport was unaltered in the period immediately following cardiac arrest. The substantial reduction in solute transport following anoxic depolarization seen at 4 min after cardiorespiratory arrest is consistent with previous reports of astrocyte swelling (Risher et al., 2009) and hindered extracellular space diffusion due to increased tortuosity (Zoremba et al., 2008). We conclude that the reduction in parenchymal tracer accumulation observed by Iliff et al. (Iliff et al., 2013) is likely due to factors other than inhibition of pulsatile flow through the interstitial space, such as reduction in ECS volume due to partial ischemia (Syková et al., 1994) or inhibition of fluid movement in the paravascular space.

The glymphatic mechanism proposes glial involvement in solute transport facilitated by AQP4; however, we found that Aqp4 deletion in mice or rats did not reduce, but perhaps slightly increased, the transfer of solutes into brain parenchyma following intracisternal injection. A small increase in parenchymal macromolecular diffusion in Aqp4-/- mice was previously found (Binder et al., 2004), which is probably due to an expanded ECS (Yao et al., 2008; Zhang and Verkman, 2010). The mildly greater solute diffusion in Aqp4-/- than in Aqp4+/+ mouse brain could account for the results here. The difference between the results here and the earlier report of Iliff et al. (Iliff et al., 2012) may be due to differences in experimental methodology or data analysis. For example, beveled glass micropipettes of ~40 μm diameter and pulsed pressure injection were used here, whereas Iliff et al. used a 30-gauge metal syringe and constant flow injection for solute application into the cisterna magna. The choice of anesthetics may have also differed between the two sets of experiments (avertin vs. unspecified). The analysis method used by Iliff et al., and reproduced here in Figure 5B for comparison purposes, is inherently non-quantitative as it is based on subjective intensity thresholding that treats large differences in fluorescence intensity, indicative of large variation in solute concentration, as equal. Finally, the experiments and their analyses here were done by an investigator blinded to genotype information. Our results also do not support a role for AQP4 in convection-driven clearance of beta amyloid, though they do not rule out the possibility that AQP4 may contribute to the pathology of Alzheimer’s disease in other ways.

In conclusion, our results do not support the glymphatic clearance mechanism proposed by Nedergaard and colleagues in which transfer of solutes from cerebrospinal to interstitial fluid requires AQP4-dependent convection in brain parenchyma. Instead, our data are consistent with modeling results demonstrating that parenchymal diffusion, coupled to convective or dispersive flow in the paravascular spaces (Asgari et al., 2016; Jin et al., 2016), is sufficient to explain movement of solutes in the brain. Further investigation of how AQP4 regulates the structure of the parenchymal and paravascular extracellular spaces is needed to clarify how its deletion or redistribution affects solute clearance from the brain. Our findings suggest the need for re-evaluation of the role AQP4 in other aspects of the glymphatic hypothesis, such as clearance of β-amyloid aggregates from the brain parenchyma and solute clearance during sleep.