To outline the CSF microcirculation, tracers were infused via the cisterna magna . Both the fixable lipophilic tracer Texas red hydrazide (TXR, 0.621 kDa) and the hydrophilic tracer tetramethylrhodamine (TMR, 3 kDa) moved rapidly through the brain along cerebral blood vessels (tracer penetration: TXR 12.57 ± 4.41 and TMR 38.71 ± 7.70% brain area at 30 min). Surprisingly, lipophilic tracers of small molecular weight showed as limited parenchymal penetration as large hydrophilic tracers (fluorescein isothiocyanate dextran, FITC, 2000 kDa) in cortical grey matter (tracer penetration 15.56 ± 2.81% brain area) 4,5
Rapid paravascular movement of lipophilic tracers.
We used in vivo
two-photon laser scanning microscopy (2PLSM) to further explore the highly selective paravascular movement of lipophilic tracers. This restricted movement is unexpected as biologically relevant lipid molecules, such as prostaglandins, cholesterol and palmitic acid, are small (< 1 kDa) and cell permeable8,9,10,11,12
. We demonstrated that the movement of small (< 1 kDa) lipophilic tracers was highly selective to the paravascular space (palmitic acid, rhod-2, TXR, sulforhodamine SR101 and Oregon green BAPTA-1 OGB) Supplementary Fig. 1a)
. Intra-arterial Texas red dextran or FITC were used to morphologically distinguish cortical surface arteries and veins as well as penetrating arterioles and venules (Supplementary Fig. 1a)4
. Cross-sectional intensity projections of penetrating arterioles confirmed paravascular tracer selectivity . By analyzing regions of interest representing the paravascular space and the surrounding tissue , we showed that the lipophilic tracers were rapidly cleared via the paravascular space without gaining access to the surrounding tissue (normalized tracer fluorescence ratio of paravascular space to surround at 60 min: OGB 3.36 ± 0.79, SR101 3.50 ± 0.88, rhod-2 4.21 ± 1.35) . Deletion of the astrocyte water channel aquaporin-4 has recently been shown to slow the circulation of hydrophilic tracers in CSF, but did not affect lipophilic tracer movement (paravascular space to surround ratio in Aqp4−/−
at 60 min: 4.83 ± 1.42)4
Lipophilic tracers selectively enter and exit brain via paravascular space surrounding arterioles and venules.
We next examined whether lipopohilic tracers enter and exit the brain via similar arterio-venous paravascular routes as hydrophilic molecules4
. Using NG2-DsRed mice that have fluorescently labeled vascular smooth muscle in arterioles, we showed that the biologically relevant tracer palmitic acid entered via a para-arterial route . Moreover, using 2PLSM imaging we showed that lipophilic tracer (rhod-2) moved sequentially in the paravascular space surrounding surface arteries, penetrating arterioles, capillaries and venules following cisterna magna infusion (normalized fluorescence of rhod-2 to eGFP expressed under the astrocyte specific Glt1
promoter: 30 min: arteriole 1.90 ± 0.38, capillary 0.45 ± 0.247, venule 0.23 ± 0.172; 60 min: arteriole 2.34 ± 0.44, capillary 0.94 ± 0.21, venule 0.44 ± 0.25; 90 min: arteriole 1.74 ± 0.32, capillary 0.72 ± 0.27, venule 1.33 ± 0.34) . These observations indicate that lipophilic molecules enter the brain via para-arterial and exit via para-venous routes.
To investigate the consequences of disrupting the paravascular microcirculation, we temporarily depressurized the CSF compartment by puncturing the cisterna magna (CMP) . Previous studies have shown that this procedure depletes ventricular and subarachnoid CSF circulation13
. Using 2PLSM to image paravascular tracer movement we demonstrate that CMP also drains all tracer from the PVS (normalized fluorescence of rhod-2 to eGFP before 4.21 ± 1.35 vs. after CMP 0.24 ± 0.15) .
Depressurizing the paravascular space impairs lipid transport and astrocyte signaling.
Since the paravascular CSF circulation appears to prevent unspecific lipid diffusion into the brain parenchyma, we next hypothesized that CMP might accelerate lipid tracer accumulation in the parenchyma. We took advantage of astrocyte specific calcium indicators (such as rhod-2), which are lipophilic tracers that become concentrated inside cells due to their acetoxymethyl group14,15
. This improved the sensitivity for detecting parenchymal influx. We found that CMP accelerates intracellular accumulation of lipophilic tracer rhod-2, when this was applied to the cortical surface or injected intraparenchymally (eGFP normalized fluorescence of rhod-2 astrocyte labeling intensity at 30 min for sham control: 1.54 ± 0.36 vs. CMP: 4.01 ± 0.57) . Conversely, Aqp4
deletion, which slows paravascular water movement, did not enhance cellular tracer uptake (Aqp4−/−
control at 30 min: 1.56 ± 0.27) . Thus, an intact paravascular space restricts lipid diffusion and cellular uptake.
To investigate the role of the paravascular space as a signaling compartment, we compared spontaneous astrocyte calcium activity in the cortex of awake mice subjected to CMP or sham surgery. Interestingly, depressurizing the paravascular space caused increased frequency and decreased synchronization of calcium signaling (ctrl 2.21 ± 0.19 vs. CMP 3.08 ± 0.29 mHz cell−1
; cell-cell correlation: ctrl 0.69 ± 0.03 vs. CMP 0.60 ± 0.03) 15
. Other aspects of astrocyte signaling were not affected (amplitude: ctrl 39.08 ± 2.10% vs. CMP 39.98 ± 2.18%; duration: ctrl 21.29 ± 1.29 s vs. CMP 24.74 ± 1.62 s; P(active over 15 min): ctrl 75.83 ± 4.20% vs. CMP 7981.05 ± 4.18%) (Supplementary Fig. 1b–d)
. Astrocyte calcium activity has been shown to propagate along blood vessels and the waves are largely ATP mediated16,17,18,19
. We therefore inserted a microelectrode into the paravascular space in situ
and stimulated calcium transients by injecting ATP. The rapid movement of agonist in the paravascular space stimulated a brisk calcium wave spreading outwards from the blood vessel, which propagated faster and over a larger area than when ATP was injected intraparenchymally (wave propagation: parenchyma 4.47 ± 0.56 vs. paravascular space 8.89 ± 1.22 μm s−1
; wave diameter: parenchyma 142.86 ± 12.50 vs. paravascular space 315.81 ± 51.42 μm) .