Interstitial fluid drainage is impaired in ischemic stroke and Alzheimer’s disease mouse models
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- Arbel-Ornath, M., Hudry, E., Eikermann-Haerter, K. et al. Acta Neuropathol (2013) 126: 353. doi:10.1007/s00401-013-1145-2
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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.
KeywordsAlzheimer’s diseaseAmyloid βCerebral amyloid angiopathyInterstitial fluidsIschemic strokePerivascular space
The brain extracellular fluids consist of blood, cerebrospinal fluid (CSF) and interstitial fluid (ISF). More is known about the CSF than the ISF with respect to its generation, composition, flow rates and paths. The ISF is thought to derive from metabolic activity of the tissue and capillary secretion of fluids and metabolites, along with some recycled CSF. Over a century of hypotheses and experiments have focused on the nature of the ISF, its origin, its relationship with the CSF and its dynamics (reviewed in ); however, these efforts mainly used indirect measures and relied on postmortem analyses.
ISF drainage is not only an important aspect of normal brain function, but is also implicated in disease, particularly the two major brain amyloid β (Aβ) amyloidoses, Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). As Aβ peptides are generated and cleared continuously in the brain, an imbalance between these two processes likely underlies this peptide’s accumulation and deposition. Late onset sporadic AD in particular, which accounts for the vast majority of cases, is thought to be associated with failure to clear the peptide from the aging brain [10, 33]. Several mechanisms mediating Aβ clearance from the brain have been described, including enzymatic degradation , transcytosis across the blood–brain barrier via the low-density lipoprotein receptor , microglia and perivascular macrophage uptake  and perivascular drainage along with the ISF . The contribution of perivascular drainage of Aβ to cerebral Aβ levels raises the possibility that vascular pathologies may interfere with this process and thus affect the disease onset and progression. Using intracerebral dye injection and post mortem examination, altered perivascular drainage of dextran was correlated with both increase in age and CAA . Additionally, we have previously shown that experimental vascular occlusion or stroke in an Aβ-expressing transgenic mouse model caused transient increases in amyloid accumulation .
Most prior studies of ISF drainage have utilized intracranial injections of soluble tracers and timed sacrifice of the animals, followed by anatomical mapping of the tracer spread. Intraparenchymal injection of soluble tracers, such as radioactive material, horseradish peroxidase, Evans blue, and India ink into different sites within rabbit and rat brains revealed a similar spread along the walls of cerebral arteries and eventual accumulation in the neck lymph nodes [4, 7, 39]. Although these past studies have contributed enormously to our understanding of ISF flow, they have been unable to define the dynamic nature of the ISF, the pathways by which it flows, and the driving forces that influence clearance rates. The major limitation of the prior approaches is that they monitor ISF flow in single postmortem snapshots rather than in real time in living animals.
In order to gain more quantitative information about the ISF flow with high spatial and temporal resolution, and to study the contribution of vascular function as well as amyloid deposition, we devised novel methods to visualize ISF drainage in the living mouse brain using multiphoton microscopy. To that end, we combined a craniotomy and laser-guided micropipette injection of fluorescent tracers into the brain parenchyma. This technique allowed us, for the first time, to watch ISF drainage in real time in mouse models of neurological disease. Our results indicate specific clearance of ISF by bulk flow along pericapillary and periarterial spaces and marked sensitivity of this process to an experimental model of stroke and to Aβ deposition.
Materials and methods
C57BL/6J males, 3–6 months of age, were obtained from Charles River Laboratories. Myh11-cre,-EGFP mice, expressing EGFP in their smooth muscle cells, were obtained from Jackson Laboratory (stock number 007742) and used at 6–12 months of age. APPswe:PS1dE9 mice (referred as APP/PS1 in the text), expressing the familial Swedish mutation in human amyloid precursor protein (APP) encoding gene and the exon 9 deletion variant of human presenilin 1 (PS1) gene both in the same locus under the control of PrP promoter , were obtained from Jackson Laboratory (stock number 00462). Mice of both genders were used either at 6–8 months of age or at 2.5–3 months of age (pre-amyloid deposition). In each genotype, mice were equally distributed according to their gender. All studies were performed with the approval of the Massachusetts General Hospital Animal Care and Use Committee and in compliance with the National Institute of Health guidelines for the use of experimental animals.
Cranial window surgery
Mice were anesthetized with isoflurane (1.5 % with pure O2) and a cranial window, 6 mm in diameter, was implanted as previously described  with minor modifications. As this procedure is followed by laser guided pressurized micropipette injection, we did not use a coverglass to close the exposed brain area. Instead, a wax ring, made of warm paraffin, was placed around the exposed brain and agarose (1.5 % in PBS) was applied to the brain surface to minimize movement artifacts (due to breathing and heart beat). PBS pH 7.4 was maintained above the agarose to keep the brain moist and enable imaging through a water immersion objective. Texas-Red labeled dextran (70 kDa, 250 μl of 12.5 mg/ml in sterile PBS, Life technologies) was injected into a lateral tail vein to provide a fluorescent angiogram.
In vivo imaging and pressure injections of fluorescent dextrans
For in vivo imaging, an Olympus FluoView FV1000MPE multiphoton laser-scanning system mounted on an Olympus BX61WI microscope and an Olympus 25× dipping objective (NA = 1.05) were used. A DeepSee Mai Tai Ti: sapphire mode-locked laser (Mai Tai; Spectra-Physics) generated two-photon excitation at 750 nm, and detectors containing three photomultiplier tubes (Hamamatsu, Ichinocho, Japan) collected emitted light in the range of 420–460, 495–540, and 575–630 nm. Mice were placed on the microscope stage and heated throughout the pipette injection and imaging session, using a heating pad with a feedback regulation from a rectal temperature probe (Harvard apparatus). Mouse anesthesia was maintained at 1–1.2 % isoflurane. A patch pipette with a tip diameter of 2–3 μm was back-filled with 3 μl of Cascade Blue labeled dextran (3 kDa, 2 mg/ml in sterile PBS; Life technologies) and inserted into the cortex to a depth of 100–150 μm from the surface using the visual guidance of Texas-Red labeled brain vasculature. Once the micropipette was placed, a z-series (508 × 508 μm, 5 μm steps, depth of about 200 μm, 512 × 512 pixels) was taken before the injection. The dextran was then pressure-ejected under laser guidance from the pipette at 5–20 PSI for 10–30 s using the Picospritzer 2000 (AM Systems, Carlsborg, WA) and the same cortical volume was imaged with 2 min time lapse imaging for 30–45 min.
In the case of APP/PS1 transgenic mice, a permanent catheter was attached to the tail vein prior to the imaging and used for injection of the BBB permeable derivative of Congo Red that binds fibrillar amyloid deposits, methoxy-XO4 (3.5 mg/Kg) , at the end of the session to visualize the Aβ aggregates on the vessel wall as well as in the parenchyma. Images of the same volume with the methoxy-XO4 positive stain were taken 30 min after the intravenous injection of the dye using the same acquisition and laser power parameters in all tested mice to allow comparison of amyloid burden across brains.
To characterize the effect of intra-peritoneal injection of phenylephrine (referred as PE in the text) on blood pressure, C57BL/6J males at 3–6 months of age were used (n = 3). For full systemic physiological monitoring, the left femoral artery was catheterized for blood sampling and measurement of mean arterial pressure (MAP; ETH 400 transducer amplifier) under isoflurane anesthesia (2.5 % induction, 1 % maintenance, in O2). Arterial blood gases and pH were measured once every 30 min during each experiment in 25 μl samples (Corning 178 blood gas/pH analyzer, Ciba Corning Diag). Mice were injected with 3 mg/Kg PE (in PBS, pH 7.4; Baxter Healthcare Corporation) and mean arterial pressure (MAP) was monitored for 1 h.
To evaluate the effect of increased peripheral blood pressure on perivascular drainage in the brain, we used a different cohort of C57BL/6J males and performed two consecutive pressure micropipette injections of Cascade Blue dextran. After the first time course, we injected through the peritoneum either PE (3 mg/Kg in PBS pH7.4, n = 6) or an equal volume of PBS (n = 7). 10 min later, the second pipette injection was performed followed by time lapse imaging with 2 min intervals.
C57BL/6J males (n = 6), 3–6 months of age, were used to evaluate the effects of microstroke on ISF drainage. To that end, the cranial window and micropipette injection were performed as described above. After the first injection and time lapse imaging, a focal stroke was induced via photothrombosis of Rose Bengal (Rose Bengal sodium salt, Sigma-Aldrich, USA). Rose Bengal was administered through a permanent intravenous catheter (100 ul, 16.6 mg/Kg in sterile PBS pH 7.4) as previously described  and immediately activated with a 543 nm HeNe laser (at maximal power) directed at the selected arterial segment (~20 × 20 μm) for 2–3 min. Segments were selected from vessels with a diameter range of 20–50 μm. RBC velocity was assessed before and after microstroke using bi-directional line scans along the length of the center of the vessels for the chosen vessel segments as well as other non-stroked segments, as previously described . The resulting line scan (LS) image appears as streaks which represent non fluorescent RBC that move through a fluorescent background. The calculated slopes of these streaks are inversely proportional to the RBC velocity. After assuring that a microstroke was indeed induced a second pressure injection of the Cascade Blue dextran was performed (using identical parameters) and the same cortical volume was imaged at 2 min intervals for an additional 30 min.
Image processing and analysis
Image processing and analysis was performed using ImageJ software (http://rsbweb.nih.gov/ij/). To reduce noise, a median filter with radius 2 was applied to all volumes. To evaluate dextran clearance rates, regions of interest showing dye localization along vessel walls were selected manually from the individual 5 μm slices, using the polygon selection tool. Dye intensity, measured in these regions of interest throughout the time course, was normalized to time zero. Curve fitting was done in MATLAB using the curve fitting tool box. We analyzed several summary measures that were independent of curve fitting, that reflect the rate of dye clearance along perivascular spaces including, time to 50 % clearance, time to 75 % clearance, area under the curve (AUC) at 30 min and AUC at 12 min (representing mostly the fast decay phase). We report results based on the measure of AUC of the intensity vs. time plots up to 30 min as it includes the entire data set obtained throughout the imaging session without the need for curve fitting. Measurements of time to 50 % and time to 75 % of initial dye intensity followed similar qualitative trends to that of AUC measurement across the different experimental groups. Thus, for the sake of simplicity, we chose to report only the AUC measurements. Area under the curve was measured using GraphPad Prism 5 software. Red blood cell (RBC) velocity was calculated from the repeated line scans as it is inversely proportional to the slope of the RBC streaks. A macro was written in Image J to detect the lines, calculate the slopes and invert the values to give the velocity. Vessel diameter was measured in Image J at three different points along the chosen vessel segment and averaged for each vessel. Arteries and veins were identified in the field of view according to their morphology and blood flow directionality. For illustration purposes, the maximal projection images of the EGFP mice were processed to remove autofluorescence lipofuscin particles.
To compare groups based on AUC, we tested the hypotheses of interest within the framework of a mixed effects model that included a random effect for mouse to adjust for correlation within mouse, and fixed effects for the comparison of interest (e.g., pre vs. post or vessel type), and in some cases for vessel diameter. Examination of residuals from the fitted model supported the assumptions of linearity and normality. For sensitivity analysis, we also tested our hypotheses using an extension of the Wilcoxon nonparametric, rank-based test to clustered data structures . The qualitative results from these analyses support those from the parametric linear mixed effects models.
Visualizing ISF drainage along perivascular spaces
To visualize ISF flow in the living mouse brain in real time, we combined laser guided bolus injections of fluorescent tracers and multiphoton time lapse imaging of cortical volumes. The tracer was Cascade Blue conjugated to 3 kDa dextran which has a molecular weight similar to that of monomeric Aβ. Intravenous injection of 70 kDa dextran conjugated to Texas Red was used to label the brain vasculature and allowed insertion of the pipette with minimal damage to the surrounding blood vessels.
Transient systemic hypertension does not affect ISF drainage
Having observed selective dye localization to arteries rather than veins, we hypothesized that arterial pulse pressures may play a major role in driving the process and thus may account for the fast decay observed. Thus, we explored whether an increase in systemic blood pressure could enhance ISF drainage. The impact of systemic blood pressure on cerebral blood flow (CBF) is substantially limited, however, by the autoregulation process in which arterial diameters change in response to blood pressure or perfusion pressure variations to maintain constant CBF . To evaluate the effect of modest change in blood pressure on ISF drainage, we used phenylephrine (PE), a selective α1-adrenergic receptor agonist, and measured ISF drainage in two consecutive bolus dye injections before and after PE administration. The administration of PE induced an increase in the mean arterial pressure (MAP, measured via a femoral artery catheter) as soon as 2 min after injection, reaching a plateau within 8–10 min after injection with an average amplitude of 25.5 ± 4.4 % increase of the initial MAP and persisting up to 1 h after injection. In a separate cohort of mice we tested the effect of PE on ISF drainage by administering either the compound (n = 6) or equivalent volume of PBS (n = 7) i.p between the two bolus dye injections. Transient systemic hypertension did not affect the drainage of ISF along perivascular spaces as evident in Fig. 3 (AUC 1st inj. vs. 2nd inj : PE: p = 0.88, control: p = 0.705). The absence of a change after PE administration suggests either that the CBF autoregulation is effective in this range of pressure changes, or that MAP plays a small role in the rate of ISF clearance along the perivascular drainage pathway.
Ischemic stroke impairs ISF drainage
Considering the mild effect of systemic hypertension on ISF drainage we set out to explore the effect of more dramatic manipulations to the brain vasculature including focal ischemic stroke and Alzheimer’s disease. Epidemiological studies have shown a strong association between vascular factors predisposing to cerebrovascular disease, stroke and AD [17, 23, 26]. We have recently shown that focal stroke can trigger accelerated amyloid deposition in mouse models of AD without changes in the levels of key players in the amyloid cascade including APP, BACE1, presenilin 1, neprilysin and Insulin degrading enzyme (IDE) . We therefore hypothesized that Aβ clearance along perivascular spaces may be impaired after stroke.
Amyloid deposition impairs ISF drainage
We have developed a novel methodology to visualize ISF drainage along perivascular spaces in real time in the living mouse brain. Fluorescent-labeled dextran injection into the brain parenchyma resulted in fast movement of the dye towards cerebral arteries and capillaries (but not veins) and subsequent clearance along perivascular spaces. The fluorescent dye accumulated along the vessel wall, occupying the perivascular space and/or perivascular parenchyma, and could be observed at lower intensities within the basement membrane between the smooth muscle cells. In the healthy mouse brain, ISF drainage occurred with a bi-exponential mode of decay that was similar among cortical vessels in the same brain as well as across brains and was independent of vessel diameter. Transient systemic hypertension within a range in which brain autoregulation is effective  did not affect this process. Conversely, focal occlusion of a single vessel segment dramatically impaired ISF drainage along this vessel with minimal effect on other vessels in the field of view. Transgenic mouse models of Alzheimer’s disease demonstrated impairment in ISF drainage along perivascular spaces in mice with amyloid deposits but not in young mice prior to the onset of amyloid deposition.
Over the past century, many studies have revealed conflicting data as to the existence of ISF drainage, the pathways by which it flows, and the nature of its sources. Although the perivascular space was once thought to be open only to the subarachnoid space and closed to the brain parenchyma [44, 48], it has been confirmed since that tracers injected into the parenchyma spread inward from the brain along perivascular spaces of arteries . Quantitative efforts combining histological and mathematical modeling showed that tracer distribution after infusion into the rat brain parenchyma exceeded the one predicted by diffusion equation in terms of both concentration of the tracer and the distance it traveled , supporting a contribution from ISF flow along perivascular spaces. In support of these findings, injecting tracers of different molecular weights into the brain parenchyma showed similar rates of clearance [1, 6], also arguing in favor of bulk flow rather than diffusion as a major component of ISF flow. These studies and others (for review see ) used injections of labeled tracers and qualitative post mortem evaluation of their distribution in the brain. To be able to appreciate ISF drainage as a dynamic process, we designed and executed a method to observe this process in real time in the intact mouse brain. The data obtained here show rapid dye accumulation along the basement membrane of arteries and capillaries, excluding veins, consistent with previous reports . The observed bi-exponential mode of decay of the tracer along these spaces argues in favor of bulk flow as a major component controlling this process rather than diffusion alone . The exclusive drainage of ISF along arterial perivascular spaces suggest that arterial pulsation may drive the fast component of the observed bi-modal decay, with diffusion potentially accounting for the slow decay. We did not, however, see major differences in clearance rates of 3 and 70 kDa dextrans (data not shown), arguing against diffusion as a major contributor for this process. In the same control experiments, a 2,000 kDa dextran did not exhibit clearance from the brain parenchyma, consistent with a physical size limit for diffusion in the extracellular space .
The dramatic impairment of ISF drainage we observed after focal vessel occlusion strengthens the contribution of cerebral perfusion as a major driving force of this process. Acute BBB breakdown and leakage of plasma proteins following a thrombotic event were previously reported [11, 34] and may account for an additional plausible mechanism for impaired ISF clearance along perivascular spaces. Changes in BBB permeability had been observed in early stages of AD . As such, serum proteins, including albumin and immunoglobulin were shown to be highly concentrated in cortical areas with dense plaque pathology. These data suggest that alterations in BBB permeability manifested chronically in AD and acutely in areas affected by stroke may lead to accumulation of plasma proteins along the vessel walls that serves as a physical impediment for clearance along these spaces.
Our data support the possibility of a direct interaction between cerebrovascular pathology, ISF flow, and Aβ deposition. Epidemiological studies have demonstrated a strong association between vascular factors predisposing to cerebrovascular disease, stroke and AD [17, 23, 26, 30] and population based neuropathological studies have shown that mixed Alzheimer’s and vascular ischemic pathologies are the major correlates of dementia in the elderly, suggesting that a link may exist between the two cerebral lesions [28, 46]. We have recently shown that focal stroke can trigger accelerated amyloid deposition in mouse models of AD . Aβ is present in the ISF  and its deposits have been observed post mortem in perivascular spaces of human AD patients . Moreover, the accumulation of Aβ within the basement membrane of cortical capillaries and arteries is very similar to that of tracers injected into either brain parenchyma [16, 45, 49] or CSF . The current findings suggest that impaired ISF drainage along perivascular spaces may be the mechanism for the observed enhancement of amyloid deposition following focal stroke . Consistent with our findings, cerebral hypoperfusion in a CAA mouse model has recently been shown to lead to accelerated accumulation of Aβ on leptomeningeal vessels .
In addition to providing a mechanism by which vascular occlusion might impede ISF clearance, the current data also point to amyloid deposition itself as a factor in perivascular drainage. As ISF clearance along perivascular spaces represents one of the routes for Aβ elimination from the brain, the expected consequence of impaired ISF drainage would be increased Aβ concentration and enhanced deposition. The observed amyloid-dependent reduction in ISF drainage might thus generate a feed-forward process by which amyloid deposition promotes more amyloid deposition. A range of structural and functional alterations in the cerebrovasculature have been reported in the aging human and mouse brain [5, 16, 22, 41]. CAA in particular has been associated with loss of smooth muscle cells, thickening of the basement membrane, narrowing of the vessel lumen, loss of cerebrovascular autoregulation, decreased vascular reactivity and increased occurrence of microscopic infarcts [35, 37, 38, 41, 42], all of which may further delay ISF drainage and thus exacerbate the progression of amyloid pathology. As discussed previously, focal ischemic insult impairs ISF drainage which in turn leads to increased Aβ deposition in the parenchyma and on vessel walls. Conversely, severe CAA is associated with increased occurrence of microinfarcts  which may further delay ISF drainage and contribute to the build-up of Aβ as well as to cognitive decline. Unlike acute focal stroke, the changes in vessel physiology during the development of CAA are progressing over a long period of time, leading to hypoperfusion and loss of cerebrovascular autoregulation, which gradually contribute to the development of the ischemic process which further exacerbates ISF drainage and the pathology in the affected areas.
Our findings are consistent with a recent report of impairments in ISF drainage in aged brains that was further aggravated in aged Tg2576 mice . ISF drainage was quantified as the number of dextran positive vessels in the brain counted post mortem and showed a lower number of dextran positive capillaries in aged Tg2576 mice, in agreement with a decreased density of capillaries in the hippocampus in the same animals. Increased numbers of dextran positive veins were also observed in the old Tg2576 mice. Our use of real-time imaging of ISF drainage for individual vessel segments has the advantage of eliminating the potential confound of variations in vessel density introduced with the postmortem approach. We also note that unlike the prior study, we did not observe dextran clearance along veins, suggesting that perivascular clearance occurs only in arteries and capillaries, which are also the vessels affected by CAA. Notably, since we used a double transgenic mouse model with accelerated amyloid deposition, our animals could be studied at 6–8 months of age, a range where vascular aging per se is minimal.
In summary, the novel method of real-time imaging described here offers insights into both the mechanisms that control solute homeostasis in the living brain and into potentially important determinants of amyloid clearance and deposition. Our findings strengthen the notion of bulk flow as the primary method for ISF clearance and suggest a major role for intravascular blood flow in driving this process. These data also link cerebrovascular risk factors to impaired ISF clearance leading to dyshomeostasis of solutes in the brain that could underlie a range of neurological disorders. Our data support a feed-forward mechanism by which CAA-related impairments in vascular function may continually promote and accelerate further Aβ deposition.
We wish to express our gratitude to Dr. K. V. Kuchibhotla, Dr. A. Serrano-Pozo and Dr. T. L. Spires-Jones for fruitful discussions and careful reading and editing of the manuscript. This work was supported by the National Institute of Health (Grant No. EB000768).