Interstitial fluid drainage is impaired in ischemic stroke and Alzheimer's disease mouse models

Abstract

The interstitial fluid (ISF) drainage pathway has been hypothesized to underlie the clearance of solutes and metabolites from the brain. Previous work has implicated the perivascular spaces along arteries as the likely route for ISF clearance; however, it has never been demonstrated directly. The accumulation of amyloid β (Aβ) peptides in brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD), and it is likely related to an imbalance between production and clearance of the peptide. Aβ drainage along perivascular spaces has been postulated to be one of the mechanisms that mediate the peptide clearance from the brain. We therefore devised a novel method to visualize solute clearance in real time in the living mouse brain using laser guided bolus dye injections and multiphoton imaging. This methodology allows high spatial and temporal resolution and revealed the kinetics of ISF clearance. We found that the ISF drains along perivascular spaces of arteries and capillaries but not veins, and its clearance exhibits a bi-exponential profile. ISF drainage requires a functional vasculature, as solute clearance decreased when perfusion was impaired. In addition, reduced solute clearance was observed in transgenic mice with significant vascular amyloid deposition; we suggest the existence of a feed-forward mechanism, by which amyloid deposition promotes further amyloid deposition. This important finding provides a mechanistic link between cerebrovascular disease and Alzheimer disease and suggests that facilitation of Aβ clearance along the perivascular pathway should be considered as a new target for therapeutic approaches to Alzheimer disease and cerebral amyloid angiopathy.

Fig. 1. Characterization of ISF drainage in the healthy mouse brain

To visualize ISF drainage in the mouse cortex, a 3kDa dextran conjugated to Cascade Blue was injected into the brain parenchyma at about 150μm depth via a thin glass pipette. A 70kDa dextran conjugated to Texas-Red was injected i.v for visualization of the brain vasculature. (a) Images represent the merged maximal projections of cortical volumes before and after the dextran injection (Texas Red in blue and Cascade Blue in red). A single channel maximal projection is shown in grey to better visualize the Cascade Blue alignment along vessel walls. The numbered white boxes indicate regions of interest (ROI) selected for quantification showed in (b). Dye alignment along vessel walls (ROI 1–4, yellow arrows) and its presence in the parenchyma (ROI 5) were followed for 30 min and quantified. (c) Dye intensity in the selected ROIs normalized to the intensity measured in the first time point (2 min post injection), shows a bi-exponential mode of decay. (d) Dye clearance along vessels from healthy brains (15 vessels from 6 different brains) shows a uniform pattern with low variability. Imaging Cascade Blue in the glass pipette above the brain surface shows no bleaching over time (light blue linear fit). (e) Area under the curve (AUC), calculated for each vessel, shows no correlation of ISF drainage with vessel diameter (0.005 ± 0.029 mean increase/μm, p=0.861). Scale bar = 100μm.

Fig. 2. Spatial distribution of dye accumulation in the brain

The localization of the dye along the vessels and within the basement membrane was investigated by performing the bolus dye injections of cascade blue dextran in mice that express EGFP in their smooth muscle cells (Myh11-cre,-EGFP mice). (a) Representative maximum intennsity projection image of cortical volumes before and soon after bolus dye injection (Individual smooth muscle cells are evident in green, cascade blue is shown in magenta). White boxes show areas that are presented in (b). Individual slices, 5μm thick, of the areas denoted in the white boxes are shown (smooth muscle cells – green, cascade blue dextran – gray). Dextran accumulation is evident along the vessel length (boxes 1 and 3) in the perivascular space and/or parenchyma as well as within the basement membrane between the smooth muscle cells, as indicated by the yellow arrows in box 2. Scale bars=50μm.

Fig. 3. Transient systemic hypertension does not affect perivascular ISF drainage

The effects of mild systemic hypertension on ISF drainage was investigated by performing two consecutive bolus dye injections, each followed by time lapse imaging and peripheral application of phenylephrine (PE), between the two injections. (a) Representative maximal projection image of cortical volumes rapidly after bolus dye injection showing the Cascade Blue Dextran (shown in red) injected in the parenchyma and the brain vasculature labeled with Texas red (shown in blue). Red boxes represent dye alignment along vessel walls that was monitored over time (b) and quantified (c) before and after peripheral injection of PE (1^st and 2^nd injections, respectively). (d). Summary of area under the curve (AUC), calculated for each vessel segment, after the 1^st and 2^nd injections in six separate PE experiments (18 vessels) and seven separate control experiments (PBS injection; 20 vessels). Values presented represent the mean and S.E.M. Post vs. pre comparisons:PE – p=0.88; controls – p=0.705. Scale bar = 100μm.

Fig. 4. Focal micro-strokes impair perivascular ISF drainage

The effects of focal stroke on ISF drainage was investigated by performing two consecutive bolus dye injections, each followed by time lapse imaging, and application of focal stroke between the two injections. (a). Maximal intensity projection images of the brain vasculature before and after the Rose Bengal-induced microstroke (‘occlusion’, marked by a red box). (b) Bi-directional line scans along the vessel length were used to measure RBC velocity in the occluded and a non-occluded vessel (1 and 2 shown in A, respectively). Presented LS show the cessation of blood flow in the occluded area with minimal effect in the non-occluded area (Ls1 and Ls2, respectively). (c) Representative images of dye alignments along vessels, from the time lapse imaging in the occluded and non-occluded vessels pre- and post-occlusion are shown. (d) Dye intensities in occluded vessel and non-occluded vessel were quantified and plotted after the first and second injections (pre and post, respectively), showing a dramatic impairment in the dye clearance along the occluded vessel. (e) Summary of the area under the curve (AUC) parameter, calculated for each vessel segment, before and after occlusion, in six separate stroke experiments (6 occluded vessels, 21 non occluded vessels) as well as seven separate control experiments (no application of stroke; 20 vessels). Values presented represent the mean and S.E.M calculated using a mixed effects regression model, with random effects for mouse to adjust for within-mouse correlation, * p<0.001. Scale bar = 100μm

Fig. 5. ISF perivascular drainage is impaired in transgenic mice with deposited plaques and CAA

The effect of amyloid deposition on ISF drainage was investigated in APP/PS1 transgenic mice and wild type littermates. (a–c) An example for dye parenchymal injection in aged transgenic mice. (a) Maximal projections of cortical volumes before the dye injections (Cascade-Blue dextran in red, Texas-red dextran in blue) and at the end of the experiment after injection of Methoxy-XO4 which labels the amyloid pathology (Methoxy-XO4 also shown in red). Numbered boxes outline areas in which dye alignment was quantified over time. (b) Single 5μm images showing dextran alignment along vessel walls corresponding to the boxes in a (yellow arrows indicate quantified areas). (c) Dye intensity decay curves fitted with bi-exponential models. (d–f) A similar experiment performed in an age-matched wild type littermate. (d) Maximal projections before and 30 min after the dye injections are shown. Numbered boxes outline areas in which dye alignment was quantified over time. (e) Single 5μm slice showing dextran alignment along vessel walls of the boxes in d (yellow arrows indicate quantified areas). (f) Dye intensity decay curves fitted with bi-exponential models. (g) AUC summary, calculated for each vessel segment from transgenic and wild-type animals at 6–8 months of age (n=8 mice/group; tg- 24 vessels; wt −28 vessels) as well as in young pre-plaque transgenic mice at 2.5–3months of age (n=5 mice, 20 vessels). Values represent the mean and S.E.M. ** p=0.007; *p= 0.011. Scale bar = 100μm.