(I-358) Mass Dependent Waste Clearance Directionality in Microfluidic Glymphatic System

Introduction:: The glymphatic theory proposes waste clearance in the brain parenchyma occurs via the convective flow across the periarterial space, brain parenchyma and perivenous space. Several in vivo studies have used intracisternal injections of fluorescent dextran of various sizes to determine the directionality of waste accumulation with contradictory results. Here, we developed a three-dimensional (3D) microfluidic in vitro model of the glymphatics (“glymphatics-on-chip”) containing two acellular “perivascular” channels and an astrocyte parenchyma embedded with 70kDa and 500kDa fluorescent dextran. Using the system, we discovered the directionality of dextran accumulation is size-dependent within the glymphatic system.

Materials and Methods:: The glymphatics-on-chip (Fig. 1A) was manufactured via soft lithography of polydimethylsiloxane (PDMS) and contains human primary astrocyte cells in a 3D hydrogel as an extracellular matrix (ECM) with embedded 70kDa Texas Red dextran and 500kDa FITC dextran between two acellular “perivascular” channels (Fig. 1B). The glymphatics-on-chip model has continuous channel flow and inducible interstitial convection from the left channel to the channel. The fluorescent dextran accumulation in each acellular channel was collected following 16 hours of induced interstitial convection and measured via plate reader.

Results, Conclusions, and Discussions:: The glymphatics-on-chip system with the two sizes of fluorescent dextran (Fig. 1A) was tested in multiple conditions with astrocytes (Fig. 1B) and without them. Without astrocytes, both sizes of fluorescent dextran accumulated evenly across the device without a specific pattern of aggregation (results not shown). However, with astrocytes, there was a statistically significant directionality for the larger (500kDa) dextran to accumulate in the right channel (Fig. 1C) while there was no such statistically significant pattern of aggregation for the smaller (70kDa) dextran (Fig. 1D). This pattern of accumulation is summarized in Figure 1E. In both sizes, more dextran was cleared in the cellular devices than the acellular controls. These results imply that while both routes of accumulation are cell-dependent, they are separate. The larger dextran appears to move via advection, in the same direction as the interstitial fluid convection. The smaller dextran may be more reliant upon diffusion since it had an equal pattern of accumulation that was not dependent on the directionality of the fluid convection. This may explain why in vivo experiments have had such discrepancies that have spawned alternative theories to the glymphatic theory, such as the intramural periarterial drainage (IPAD) theory.

In this study, a glymphatics-on-chip model demonstrated that the directionality of waste clearance and accumulation is mass-dependent, matching both the perivenous accumulation pattern of glymphatic theory and the periarterial accumulation of the IPAD theory with a single defined convection direction. This insight is integral for understanding the healthy physiology of the glymphatic system and sheds light upon the apparent discrepancy in aggregation directionality found in existing in vivo experiments. This system can further be used to discern the mechanisms behind these two separate patterns of waste clearance and accumulation in future studies.


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