Huber VJ et al. (2018), Aquaporin-4 facilitator TGN-073 promotes inters...

XB-ART-55889

Neuroreport 2018 Jun 13;299:697-703. doi: 10.1097/WNR.0000000000000990.

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Aquaporin-4 facilitator TGN-073 promotes interstitial fluid circulation within the blood-brain barrier: [17O]H2O JJVCPE MRI study.

Huber VJ , Igarashi H , Ueki S , Kwee IL , Nakada T .

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The blood-brain barrier (BBB), which imposes significant water permeability restriction, effectively isolates the brain from the systemic circulation. Seemingly paradoxical, the abundance of aquaporin-4 (AQP-4) on the inside of the BBB strongly indicates the presence of unique water dynamics essential for brain function. On the basis of the highly specific localization of AQP-4, namely, astrocyte end feet at the glia limitans externa and pericapillary Virchow-Robin space, we hypothesized that the AQP-4 system serves as an interstitial fluid circulator, moving interstitial fluid from the glia limitans externa to pericapillary Virchow-Robin space to ensure proper glymphatic flow draining into the cerebrospinal fluid. The hypothesis was tested directly using the AQP-4 facilitator TGN-073 developed in our laboratory, and [O]H2O JJ vicinal coupling proton exchange MRI, a method capable of tracing water molecules delivered into the blood circulation. The results unambiguously showed that facilitation of AQP-4 by TGN-073 increased turnover of interstitial fluid through the system, resulting in a significant reduction in [O]H2O contents of cortex with normal flux into the cerebrospinal fluid. The study further suggested that in addition to providing the necessary water for proper glymphatic flow, the AQP-4 system produces a water gradient within the interstitial space promoting circulation of interstitial fluid within the BBB.

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Species referenced: Xenopus laevis
Genes referenced: aqp4 cldn2
GO keywords: circulatory system process [+]

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	Fig. 1. Schematic presentation of polarized localization of aquaporin-4 (AQP-4). (a) Relationship with Virchowâ€“Robin space (VRS). Expression of AQP-4 in the brain is highly polarized to end feet of astrocytes at two specific locations: the glia limitans externa (GLE) at the cortical surface and pericapillary VRS. VRS constitutes fluid-filled canals surrounding perforating arteries and veins in the parenchyma of the brain. Although the pia mater ends near the brain surface, VRS continues into the brain parenchyma with a perforating artery. The arterial wall is surrounded by smooth muscle, which plays a main role in controlling capillary inner pressure (autoregulation), whereas the capillary is surrounded by interstitial fluid, the hydrodynamics of which are controlled by influx of water from intracellular fluid of astrocytes through AQP-4. (b) Astrocyte and end feet. Astrocyte end feet attach to many structures, but AQP-4 is found only at the GLE and the VRS. As AQP-4 at the capillary VRS is responsible for water efflux from astrocytes into VRS, it is highly plausible that AQP-4 at the GLE is responsible for water influx into the astrocyte from pericortical interstitial fluid space, thereby maintaining astrocyte intracellular water equilibrium. Diagram modified from Hirano 11 and Sasaki and Mannen 12, which show that a single astrocyte projects end feet both at the GLE and VRS 11,12. Similar to the case of potassium siphoning, water homeostasis can be performed efficiently by a single astrocyte or may require a gliaâ€“cell syncytium 11,13.
	Fig. 2. Schematic presentation of the aquaporin-4 (AQP-4) system. The AQP-4 system provides additional water flow into the pericapillary Virchowâ€“Robin space (VRS). Necessary water enters astrocytes through AQP-4 at the glia limitans externa (GLE). This internal facilitator promotes appropriate interstitial fluid dynamics including flow through the VRS (interstitial flow), which constitutes glymphatic flow. This model is readily examined in-vivo by analyzing tracer dynamics injected into the systemic vein. Broken line squares indicate the components examined in the in-vivo dynamic studies using [17O]H2O JJ vicinal coupling proton exchange (JJVCPE) imaging and the AQP-4 facilitator, TGN-073 (Fig. 6).
	Fig. 3. TGN-073 reaction schema.
	Fig. 4. Schematic presentation of JJ vicinal coupling proton exchange (JJVCPE) imaging. The nuclear magnetic resonance (NMR)-sensitive, nonradioactive isotope of oxygen, oxygen-17 [17O], and adjacent proton will show JJ vicinal coupling. In water, the protons of the water molecule and ionized proton of the dissolved molecule can exchange among each other. Accordingly, appropriately designed [17O]-labeled molecules can alter the apparent T2 of water molecules under NMR experiments. Using T2-weighted imaging, this can be developed into noninvasive imaging, JJVCPE imaging that is capable of quantifying the contents of the target molecules, akin to radioactive tracer imaging such as PET 9,25.Signal intensity change, Î´S, of the voxel with [17O]-labeled substrate can be given by:where S0 is the original signal intensity, TE is the echo time, Ï is the relative concentration of the [17O]-labeled substrate, and Ï is the proton exchange rate. Although it is difficult to determine the absolute concentration of the [17O]-labeled target molecule with this imaging method, it is still possible to obtain dynamic data for a target molecule in space and time, given the high spatial resolution inherent to MRI. In contrast to radiotracer methods, tracer ([17O]-labeled target molecule) contents have an inversed correlation with signal intensities, namely, higher tracer contents yield lower signal intensities.
	Fig. 5. JJVCPE data. (a) ROI and decay curve fitting. Upper: scout film showing regions of interest (ROI). Imaging slab was set to 6â€‰mm caudal from the top of the cerebrum (left) and ROI was selected semiautomatically using image processing software. Lower: Decay curve fitting. I0 shows the normalized signal intensity at infinite time (t=âˆž) calculated from the fitted curve. As described, higher tracer contents will yield lower I0. (b) Representative time course. Representative time curve of signal intensities within pixels of each ROI shown in (a) following intravenous (i.v.) [17O]H2O administration in control mouse. Blue: cortex, red: basal ganglia (BG), green: cerebrospinal fluid (CSF) within the third ventricle. Each dot represents the intensity of each pixel within the ROI.
	Fig. 6. Xenopus laevis oocyte bioassay. (a) Time-dependent volume change plots are shown for water-injected oocytes incubated for 30â€‰min before initiation of hypotonic shock with a blank (black circle), and AQP-4 cRNA-injected oocytes incubated with a blank (red square) or TGN-073 (blue triangle). (b) Hypotonic flux (Pf) of the sham, blank, and TGN-073 groups is shown as black bars plus SEM. Statistical significance between the blank and TGN-073 groups is indicated by **P=0.0025 (one-way analysis of variance with Fisherâ€™s least significant difference test).
	Fig. 7. Group analysis of TGN-073 effect. Group treated with an experimental dose of TGN-073 (200â€‰mg/kg) showed significantly higher I0 in the cortex compared with the saline-treated group, indicating higher turnover of [17O]H2O in the cortex. The group treated with a control dose of TGN-073 (20â€‰mg/kg) did not show any significant effects, excluding potential nonspecific effects of TGN-073. *P=0.0066 (t-test). BG, basal ganglia; CSF, cerebrospinal fluid.

References [+] :

Abbott, Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology. 2004, Pubmed