Research ArticleEYE PHYSIOLOGY

An ocular glymphatic clearance system removes β-amyloid from the rodent eye

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Science Translational Medicine  25 Mar 2020:
Vol. 12, Issue 536, eaaw3210
DOI: 10.1126/scitranslmed.aaw3210
  • Fig. 1 Existence of an ocular glymphatic clearance system.

    (A) Top: Schematic of intravitreal injection of hAβ. Insert: IOP during injection (n = 5 to 6, P = 0.0516 to 0.543, unpaired two-tailed t test). Rectangle indicates proximal optic nerve displayed in (B to J). Bottom: uDISCO-cleared transparent mouse heads 1 hour after hAβ injection. (B) Confocal images of ipsilateral retina (left) and optic nerve (right) after hAβ intravitreal injection. (B) and (D) inserts display macroscopic images of the eye and optic nerve injected with respective tracers without background subtraction. (C) Confocal imaging and quantification of optic nerve from reporter mouse with DsRed-tagged mural cells (vascular smooth muscle cells and pericytes) 30 min after intravitreal hAβ injection (means ± SEM, n = 12 to 18). (D) Confocal image of mouse optic nerve 30 min after intravitreal AF-dextran injection. (E and F) Confocal imaging and quantification of optic nerve colabeling with TUJ1 after tracer administration (n = 9 to 11, ****P < 0.0001 unpaired, two-tailed t test). Note tracer accumulation in the dural lining of the nerve. (G) Cervical lymph nodes exhibiting intense hAβ labeling 3 hours after injection. (H) Schematic of the double injections. (I) Representative image and quantification of double injections of hAβ intravitreally and fluorescent tracer intercisternally (means ± SEM, n = 12). (J) Confocal imaging of optic nerve from reporter mouse with DsRed-tagged mural cells (vascular smooth muscle cells and pericytes) after intracisternal dextran injection with line scan quantified in (K) (means ± SEM, n = 8 to 9). Scale bars, 500 μm [A and B (right) and D, G, and I] and 50 μm [B (left) and C, E, F, and J]. A.U., arbitrary units.

  • Fig. 2 Ocular glymphatic clearance is AQP4 dependent.

    (A) Top: Experimental setup comparing tracer clearance from the retina and optic nerve after intravitreal injection in Aqp4+/+ and Aqp4−/− mice. Bottom: Scatter plot with means ± SEM overlaid comparing intraocular pressure (IOP) in Aqp4+/+ and Aqp4−/− mice before the injection. (B) Representative transverse sections of retina collected 30 min after intravitreal hAβ injection and counter-stained for AQP4. (C) Line graph overlaid on retinal illustration (top) and bar graphs (bottom) comparing hAβ tracer penetration (n = 7 to 9, *P < 0.05, Mann-Whitney test) into the various retinal layers (n = 7 to 9, **P < 0.01 and ****P < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test). n.s., not significant. (D) Representative background-subtracted heat maps of hAβ signal in the optic nerves of Aqp4+/+ and Aqp4−/− mice 30 min after intravitreal injection. (E) Top: Averaged fluorescent intensity profiles of hAβ in the optic nerves from the two groups. Bottom: The distance of tracer transport (n = 23 to 24, *P < 0.05, Mann-Whitney test). (F and G) Total hAβ signal and peak intensity in the optic nerve 30 min after intravitreal injection (n = 23 to 24, *P < 0.05, unpaired two-tailed t test for total signal and Mann-Whitney test for peak). Scale bars, 50 μm (B) and 100 μm (D).

  • Fig. 3 The TPD drives ocular glymphatic outflow.

    (A) Schematic of the setup used for analyzing hAβ transport after intravitreal injection while manipulating ICP. Top: Mean ICP and IOP normalized to control (±SEM) plotted as a function of time in the high, normal, and low ICP groups (n = 10 to 12). (B) Representative background-subtracted heat maps of hAβ in the optic nerve from high, normal, and low ICP groups. (C) Top: Averaged fluorescent intensity profiles of hAβ in the optic nerves from the three groups. Bottom: The distance of tracer transport (n = 6 to 8, ***P < 0.001 and n.s., P = 0.9976, one-way ANOVA followed by Tukey’s post hoc test). (D and E) Total hAβ signal and peak intensities in the optic nerve 30 min after intravitreal injection (n = 6 to 8 for each group, *P < 0.05; **P < 0.01; ****P < 0.0001; and n.s., P = 0.2996, one-way ANOVA followed by Tukey’s post hoc test). Scale bar, 500 μm (B).

  • Fig. 4 Light stimulation enhances hAβ along the optic nerve.

    (A) Schematic of the experimental groups. The first group was kept in darkness. The second group was exposed to 1 Hz light stimulation (100 ms duration, five lumens). The third group was pretreated with atropine (1%) before exposure to 1 Hz light stimulation. The fourth group was pretreated with pilocarpine (2%) and kept in darkness. (B) Representative background-subtracted heat maps of optic nerves from the four groups 30 min after injection of hAβ and a postmortem group 120 min after injection of hAβ. (C) Averaged fluorescent intensity profiles of optic nerves from the four groups (n = 6 to 19). (D) hAβ signal mapped as total signal, peak intensity, and distance of the hAβ transport (n = 6 to 19, *P < 0.05; **P < 0.01; ***P < 0.001; and n.s., P = 0.0756, one-way ANOVA followed by Dunnett’s post hoc test). (E) Infrared pupillometry tracking of the pupil size and light-induced constriction with and without atropine pretreatment. The pupil area (mm2) was determined by auto-thresholding. (F) Representative pupillometry recordings in dark-exposed and light-stimulated mice with and without atropine administration. (G) Left: Comparison of the variance of pupil area in these groups (n = 3, **P < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison). Middle: Spectral analysis of pupil response in these groups calculated as percentage of 1 Hz band (n = 3 to 6, *P < 0.05, one-way ANOVA followed by Dunnett’s multiple comparison). Right: Cumulative pupil diameter change over the time of experiment in these groups (n = 3 to 6, *P < 0.05, Kruskall-Wallis test followed by Dunnett’s multiple comparison). Scale bars, 500 μm (B and E).

  • Fig. 5 Disruption of the lamina barrier in two distinct murine models of glaucoma reveals a redirection and pathological enhancement of ocular glymphatic outflow.

    (A) Schematic of disease progression in the DBA/2J strain and chronic CLS model (CD-1) (44, 46). (B) Representative background-subtracted heat maps of optic nerves 30 min after hAβ injection in young, middle-aged, and old DBA/2J mice and old D2-control mice, as well as CD-1 CLS and CD-1 control mice. (C) Averaged fluorescent intensity profile of hAβ distribution along the optic nerve in old DBA/2J or CD-1 CLS compared to respective controls (n = 6 to 11). (D) Total hAβ signal in old DBA/2J or CD-1 CLS compared to respective controls (n = 6 to 11, *P < 0.05, Kruskall-Wallis followed by Dunn’s post hoc test for DBA/2J model and unpaired two-tailed t test for CLS model). (E) Representative background-subtracted heat maps of optic nerves from old DBA/2J and D2-control mice 30 min after intravitreal administration of AF-dextran. (F) Averaged fluorescent intensity profile of AF-dextran along the optic nerve in old DBA/2J or CD-1 CLS compared to respective controls (n = 6 to 9). (G) Total signal of different sized AF-dextrans in optic nerve from old DBA/2J or CD-1 CLS compared to respective controls (n = 4 to 10, *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired two-tailed t test or Mann-Whitney test). (H) Total hAβ or AF-dextran signal in the optic nerves of old DBA/2J mice plotted as a function of RGC density in their retinas (n = 6 to 8). (I) Electron micrographs of the glial lamina region from young D2-control and old DBA/2J mice. (J) Schematic of real-time TMA+ iontophoresis measurement. (K) TMA+ measurements of α (extracellular volume space) and λ (extracellular tortuosity) (n = 6 to 20, P = 0.765 for α and P = 0.177 for λ, unpaired two-tailed t test). Scale bars, 500 μm (B and E) and 0.5 μm (I).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/536/eaaw3210/DC1

    Fig. S1. Characterization of additional tracers and tracer delivery routes.

    Fig. S2. Brief retinal exposure to trace amounts of hAβ using our intravitreal infusion paradigm does not induce gliosis, microglial activation, apoptosis or BRB disruption.

    Fig. S3. Detailed analysis of ocular glymphatic clearance route.

    Fig. S4. Validation of tracer delivery model in another species and with different infusion parameters.

    Fig. S5. Light stimulation accelerates hAβ tracer movement along the optic nerve.

    Fig. S6. Influence of the translaminar pressure difference on stimulated and unstimulated tracer movement.

    Fig. S7. Tracer efflux pattern in murine glaucoma model indicates leaky lamina that may allow bypass of intra-axonal transport via extracellular route.

    Fig. S8. Murine glaucoma models reveal defects in the lamina barrier allowing escape of large intraocular tracers.

    Fig. S9. Schematic model of the ocular glymphatic clearance system and its dysfunction in a murine model of glaucoma.

    Table S1. Retinal cytokine assay comparing intravitreal hAβ injection to vehicle.

    Table S2. Comparison of the controls for the DBA/2J strain.

    Table S3. Tracers used and their routes of administration.

    Movie S1. Animation of the proposed mechanism for ocular glymphatic clearance in the healthy optic nerve and glaucoma.

    Data file S1. Tabular summary of all the data (provided as separate excel file).

  • The PDF file includes:

    • Fig. S1. Characterization of additional tracers and tracer delivery routes.
    • Fig. S2. Brief retinal exposure to trace amounts of hAβ using our intravitreal infusion paradigm does not induce gliosis, microglial activation, apoptosis or BRB disruption.
    • Fig. S3. Detailed analysis of ocular glymphatic clearance route.
    • Fig. S4. Validation of tracer delivery model in another species and with different infusion parameters.
    • Fig. S5. Light stimulation accelerates hAβ tracer movement along the optic nerve.
    • Fig. S6. Influence of the translaminar pressure difference on stimulated and unstimulated tracer movement.
    • Fig. S7. Tracer efflux pattern in murine glaucoma model indicates leaky lamina that may allow bypass of intra-axonal transport via extracellular route.
    • Fig. S8. Murine glaucoma models reveal defects in the lamina barrier allowing escape of large intraocular tracers.
    • Fig. S9. Schematic model of the ocular glymphatic clearance system and its dysfunction in a murine model of glaucoma.
    • Table S1. Retinal cytokine assay comparing intravitreal hAβ injection to vehicle.
    • Table S2. Comparison of the controls for the DBA/2J strain.
    • Table S3. Tracers used and their routes of administration.
    • Legend for movie S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Animation of the proposed mechanism for ocular glymphatic clearance in the healthy optic nerve and glaucoma.
    • Data file S1. Tabular summary of all the data (provided as separate excel file).

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