Author response: Aquaporin-4-dependent glymphatic solute transport in the rodent brain

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-β. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures. https://doi.org/10.7554/eLife.40070.001 Introduction A brain-wide fluid transport pathway, known as the glymphatic system, supports the rapid exchange of cerebrospinal fluid (CSF) and interstitial fluid (ISF) along perivascular pathways (Iliff et al., 2012). The glymphatic system consists of three principal sequential anatomic segments: (i) CSF inflow along the perivascular spaces surrounding penetrating arteries, (ii) dispersion of CSF through the wider interstitium, and (iii) efflux of ISF along the large-caliber draining veins to re-enter the CSF within the ventricular and cisternal compartments (Jessen et al., 2015). Ultimately, interstitial solutes cleared to the CSF exit the brain through meningeal lymphatic vessels flanking the venous sinuses, along cranial and spinal nerve sheathes, and across the cribriform plate (Louveau et al., 2015; Aspelund et al., 2015). Astrocytic endfeet ensheath the cerebral vasculature, and the abundantly expressed astroglial water channel aquaporin-4 (AQP4) localizes primarily to the perivascular endfoot membrane domain abutting the basal lamina. As this anatomic arrangement provides a route for rapid water movement between the perivascular space and the glial syncytium, AQP4 has been proposed to support perivascular fluid and solute movement along the glymphatic system (Nedergaard, 2013). Several groups have independently shown that the astrocytic AQP4 is essential for fast glymphatic transport. Iliff et al. demonstrated a significant suppression of both perivascular CSF tracer influx and interstitial mannitol and amyloid-β (Aβ) clearance in Aqp4 knockout (KO) mice (Iliff et al., 2012). Subsequent work demonstrated that Aqp4 gene deletion exacerbated glymphatic pathway dysfunction after traumatic brain injury (TBI) and promoted the development of neurofibrillary pathology and neurodegeneration in the post-traumatic brain (Iliff et al., 2014). Plog et al. similarly found that Aqp4 KO mice exhibit slowed transport of interstitial solutes to the blood after TBI, reflected by a significant reduction in plasma biomarkers of TBI, including GFAP, neuron-specific enolase, and S100β (Plog et al., 2015). Xu et al. reported that deletion of Aqp4 exacerbated Aβ plaque accumulation and cerebral amyloid angiopathy in the APP/PS1 murine model of Alzheimer's disease (Xu et al., 2015). Achariyar et al. found significantly reduced distribution of FITC-ApoE3, 125I-apoE2, 125I-apoE3, 125I-apoE4, as well as 14C-inulin in Aqp4 KO mice following tracer injection to the CSF (Achariyar et al., 2016). Lundgaard et al. reported that glymphatic clearance of lactate was reduced in Aqp4 KO mice (Lundgaard, 2016). Finally, Murlidharan et al. demonstrated that Aqp4 KO mice exhibit significantly impaired clearance of adeno-associated viruses (AAV) infused into the ventricles, and concluded that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS (Murlidharan et al., 2016). A recent MRI study showed the AQP4 facilitator, TGN-073, potentiated the transport of interstitial fluid from the glia limitans externa to pericapillary Virchow-Robin space (Huber et al., 2018). The critical role of AQP4 in supporting perivascular CSF-ISF exchange was recently questioned in a report by Smith et al. (2017), in which the authors failed to detect any reduction in CSF tracer influx into the brain parenchyma of Aqp4 KO mice compared to wild-type (WT) controls. Because a key element of the glymphatic hypothesis is the role of astroglial water transport in supporting perivascular CSF-ISF exchange, we consider it critical to re-examine the role of AQP4 in this process, with an aim to resolve the discrepant reports. Using data generated from five independent laboratories, we have undertaken such a re-evaluation of the effects of Aqp4 deletion on perivascular glymphatic exchange. Results of this analysis consistently confirm that Aqp4 deletion impaired perivascular glymphatic flow relative to that in wild-type mice. Our conclusion is strengthened by the use of four independently generated Aqp4 KO lines, including the line used by Smith et al. (2017), as well as the α-syntrophin (Snta1) KO line, which lacks AQP4 perivascular localization despite normal expression levels (Amiry-Moghaddam et al., 2003). Smith et al. (2017) also questioned the existence of tissue bulk flow based injecting tracers of varying molecular sizes into cortex or striatum. We questioned this approach based on the prior finding that traumatic brain injury is linked to an immediated and sustained reduction in CSF influx (Iliff et al., 2014). As expected, our analysis showed that insertion of glass pipettes into the brain markedly reduced brain-wide CSF tracer influx. Thus, interstitial fluid transport should not be studied following invasive procedures in the brain. Results The study included data from five laboratories using four independently generated Aqp4 KO lines and one Snta1 KO mouse line (Figure 1a–e) (Fan et al., 2005; Ikeshima-Kataoka et al., 2013; Ma et al., 1997; Thrane et al., 2011; Adams et al., 2000). Immunohistochemistry done in parallel with the glymphatic experiments verified that AQP4 was indeed deleted in all the Aqp4 KO mouse lines (Figure 1a–c and e). In the Snta1 KO mice, immunofluorescence demonstrated that perivascular AQP4 polarization was absent in this line (Figure 1d). Figure 1 Download asset Open asset Strategy used for generation of KO mice and the experimental design of the study. Top row of each panel represents the institution where the line originated from and the strategy used to generate the four global Aqp4 KO mice and the Snta1 KO mice. Second row of each panel shows the five labs that collected data on glymphatic system function in the five transgenic mouse lines: (a) Nanjing Medical University (NMU), (b) RIKEN Center for Brain Science (RIKEN), (c) University of North Carolina (UNC), (d) Oregon Health and Science University (OHSU), (e) University of Rochester Medical Center (URMC). (a-e) Immunohistochemical analysis showing the lack of AQP4 expression in the global KOs and (d) lack of perivascular AQP4 localization in Snta1 KO compared to WT mice. Scale bar: 50 µm. (a-e) Third row displays the volume and rate used for the intracisterna magna (CM) injections for each experiment, the tracer used, and the experiment duration. Note that URMC collected two full data sets using an injection rate of either 1 or 2 µl/min. (a-e) The last row display the analyses strategy employed by each of the five research group in Figures 2–6. (f) Additional experiments performed in Figure 7 tested the effect of intrastriatal (IS) injection on global glymphatic function. TxRd, Texas Red; BDA, biotinylated dextran amine; BSA-647, bovine serum albumin-Alexa Fluor 647; DCE-MRI, dynamic contrast-enhanced magnetic resonance imaging. https://doi.org/10.7554/eLife.40070.002 Figure 2 Download asset Open asset NMU: Aqp4 gene deletion reduced the penetration of intracisternally injected tracer into the brain parenchyma. Texas Red-conjugated dextran (TRd3, 3kD) was injected intracisternally into WT and Aqp4 KO mice. Thirty minutes after injection, the anesthetized animals were perfusion fixed, and the fluorescence was evaluated ex vivo. (a) Representative near infrared (NIR) fluorescence images of the dorsal and ventral whole-brains of four mice per genotype. (b-c) Quantification of the mean integrated optical density (MIOD) of TRd3 on the dorsal (b) and ventral (c) brain surface of WT (grey) and Aqp4 KO (purple) mice from 4.0 mm anterior to 8.0 mm posterior to bregma. (d) Representative images of coronal brain sections at +0.5 mm from bregma from five pairs of WT and Aqp4 KO mice showing TRd3 distribution within the brain. (e) High magnification micrographs of the hypothalamus (lined area in d) showing the fluorescence intensity of TRd3 within the perivascular space (star) and adjacent brain parenchyma (dotted line) of WT mice and Aqp4 KO mice, respectively. (f) Quantification of the percentage area of whole-slice fluorescence of the both genotypes for 6–8 forebrain sections (+1.7 to −0.7 mm from bregma) of each mouse. (g) Diagram showing the subregional analysis of brain sections at the level of 0.5 mm anterior to bregma. (h) Quantification of the mean fluorescence intensity (AU, arbitrary units) of TRd3 of the dorsal, ventral and lateral brain regions, respectively. (i-j) Quantification of the mean fluorescence intensity of TRd3 along the perivascular space and the interstitium adjacent to the vessels under the ventral surface of the hypothalamus of the both genotypes. Shades and error bars represent standard deviation. Data in Figure 2b and c were analyzed by repeated-measures ANOVA, N = 4 per group. Data in Figure 2f,h,i and j were analyzed by Student's t-test. p-Values shown are comparisons between WT and Aqp4-/-. ns: not significant (Figure 2—source data 1). https://doi.org/10.7554/eLife.40070.003 Figure 2—source data 1 Source data for Figure 2. https://doi.org/10.7554/eLife.40070.004 Download elife-40070-fig2-data1-v4.xlsx Figure 3 Download asset Open asset RIKEN: Aqp4 -/- mice display compromised CSF tracer infiltration under ketamine-xylazine anesthesia. (a) Schematic diagram for CM injection of BDA tracer (left) and experiment schedule (right). (b) Examples of SA-enhanced BDA distribution 30 min after CM injection. Slices at an anterior-posterior position of bregma are presented for WT (upper) and Aqp4-/- (lower) mice. Depth profile is calculated for the cortical position 3 mm lateral to the midline. (c) Mean depth profiles of SA-enhanced BDA signals for WT (black, N = 4) and Aqp4-/- (green, N = 9) mice. (d) Mean SA-enhanced BDA signal intensities (3 mm lateral to the midline, depths 0–800 µm) along anterior-posterior positions for WT (black) and Aqp4-/- (green) mice. Shades and error bars represent SEM. *p < 0.05, **p < 0.01, t-test (Figure 3—source data 1). https://doi.org/10.7554/eLife.40070.005 Figure 3—source data 1 Source data for Figure 3. https://doi.org/10.7554/eLife.40070.006 Download elife-40070-fig3-data1-v4.xlsx Figure 4 Download asset Open asset UNC: CSF tracer influx is decreased in Aqp4 KO mice. (a) Coronal sections from a C57BL/6 wild-type mouse (WT), CD1 background strain control (Aqp4+/+), and Aqp4 KO mice (Aqp4-/-) showing a fluorescent CSF tracer, BSA-647 and co-labeling with DAPI. Scale bar: 1 mm (b) Mean pixel intensity in arbitrary units (A.U.) for six brain sections of each mouse for all three groups. n = 3 (WT), 7 (Aqp4+/+), 6 (Aqp4-/-). One-way ANOVA Tukey's multiple comparisons test, Interaction term: p = 0.0110, F = 6.512, ns: not significant. (c) Diagram showing the anterior-posterior range of the quantified coronal sections relative to bregma from (b). (d) Quantification of the slices shown in (c) + 1.2 to −1.8 mm from bregma. Repeated measures two-way ANOVA with Tukey's multiple comparisons test, Interaction term: p = 0.038, F = 2.085, p values shown are comparisons of WT and Aqp4+/+ vs. Aqp4-/-. (e) Diagram depicting the ROIs included in the regional analysis of brain slices at +0.6 mm from bregma. CPu: caudoputamen; HT: hypothalamus; BF: basal forebrain; DC: dorsal cortex; LC: lateral cortex; VC: ventral cortex. (f) Mean pixel intensity of brain regions shown in (e) for coronal sections + 0.6 mm from bregma. Repeated measures two-way ANOVA Tukey's multiple comparisons test, Interaction term: p < 0.0001, F = 8.109. Data is presented as mean ±SEM (Figure 4—source data 1). https://doi.org/10.7554/eLife.40070.007 Figure 4—source data 1 Source data for Figure 4. https://doi.org/10.7554/eLife.40070.008 Download elife-40070-fig4-data1-v4.xlsx Figure 5 Download asset Open asset URMC: Glymphatic influx of CSF tracer is facilitated by AQP4. (a) Representative images from an in vivo transcranial optical imaging experiment of a WT control mice (Aqp4+/+) and Aqp4 KO (Aqp4-/-) mice starting at 15 min after intracisternal delivery of 10 µl (2 µl/min) of a 66 kDa fluorescent tracer, BSA-647. Scale bar: 2 mm. (b) Mean pixel intensity (MPI) of BSA-647 over a 30 min experiment, imaging was started at the beginning of the injection. Two-way repeated measures ANOVA with Sidak's multiple comparisons test, overall model *p = 0.0329, multiple comparisons *p < 0.05; n = 6–7/group. (c) Coronal sections collected 30 min after intracisternal injection of two varying molecular size tracers, 3 kDa Texas Red dextran and BSA-647. Mean pixel intensity for a (d) 3 kDa dextran and (e) BSA from six coronal sections between +1.2 and −1.8 mm from bregma for each animal. Two-tailed unpaired t-test, **p = 0.0097, ****p < 0.0001; n = 6–8/group. (f) Coronal section from an Aqp4+/+ mouse stained and imaged for AQP4 (magenta) and BSA-647 tracer (cyan). (g) Mean pixel intensity from a replicate experiment quantified the same as (e). Two-tailed unpaired t-test, *p = 0.0335, n = 4–6/group. (h) Mean tracer penetration depth profiles normalized to the fluorescence at the pial surface of a coronal section at bregma from the set of experiments displayed in (g). The line was placed orthogonal to the cortical surface at the most dorsal position where tracer could be found at the pial surface (f). Tracer depth in the WT mice was measured at the same position as the KO mouse. Two-way repeated measures ANOVA with Sidak's multiple comparisons test, overall model ****p < 0.0001, multiple comparisons ****p < 0.0001, n = 4–6/group. (i) Cortical depth at which the fluorescence at the surface decreases by half for the profiles in (h). Two-tailed unpaired t-test, *p = 0.0161, n = 4–6/group. (j) Relative copy numbers (RCN) of the Aqp4 gene locus and the excision sequence (k) showing successful deletion of Aqp4 exon 1–3. RCN was quantified by qPCR for mice used in the experiment in (g). Data expressed as mean ±SEM (Figure 5—source data 1). https://doi.org/10.7554/eLife.40070.009 Figure 5—source data 1 Source data for Figure 5. https://doi.org/10.7554/eLife.40070.010 Download elife-40070-fig5-data1-v4.xlsx Figure 6 Download asset Open asset Deletion of the adapter protein α-syntrophin impairs AQP4 perivascular localization, and CSF influx into the brain parenchyma. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was acquired on a 11.75 preclinical MRI scanner, and was used to characterize the effect of α-syntrophin deletion on gaditeridol influx into the brain. Representative images of AQP4 perivascular localization in wild-type mice (a), and the loss of perivascular localization of AQP4 seen in the Snta1-/- mice (b). Scale bar: 50 µm, inset scale bar: 10 µm. (c-h) Coronal slice of T1-weighted images acquired by DCE-MRI demonstrate the reduced influx of gaditeridol contrast agent into the parenchyma in Snta1-/- mice relative to wild-type mice at 30 and 60 min. Scale bar: 1 mm. (i-n) Quantification of T1 weighted signal in various brain subregions normalized to baseline at each time point. Traces for each individual animal are presented (lines) along with the summary statistics (mean ±SEM, two-way ANOVA). WT n = 5, ASYNKO n = 7. CTx = cortex (p = 0.0035) Hip = hippocampus (p = 0.0003) Subcortical = subcortical regions (p = 0.0185) 3V = 3rd Ventricle (p = 0.0284) Total (p = 0.0085) (Figure 6—source data 1). https://doi.org/10.7554/eLife.40070.011 Figure 6—source data 1 Source data for Figure 6. https://doi.org/10.7554/eLife.40070.012 Download elife-40070-fig6-data1-v4.xlsx Figure 7 Download asset Open asset A unilateral intrastriatal injection reduces global glymphatic function. (a) KX-anesthesized mice received an injection into striatum. After injection, the glass capillary was slowly removed and the skull sealed with silicone elastomer. One hour later, mice received an intracisternal injection of a 66 kDa BSA-647 tracer and brains were removed and drop fixed 60 min later. (b) Control mice only received the intracisternal injection but not the intrastriatal injection. Mice that received an intrastriatal injection were injected with either HiLyte 488-amyloid-β1-40 or an aCSF sham. (c) Global glymphatic tracer influx was quantified from a total of six coronal sections between +1.2 and −1.8 mm from bregma for each animal. Ordinary one-way ANOVA posthoc Tukey's multiple comparisons test, Control vs. Aβ-injected: ***p = 0.0006; Control vs. Sham: ***p = 0.0001; n = 6–8/group (Figure 7—source data 1). https://doi.org/10.7554/eLife.40070.013 Figure 7—source data 1 Source data for Figure 7. https://doi.org/10.7554/eLife.40070.014 Download elife-40070-fig7-data1-v4.xlsx NMU: reduced influx of a fluorescent CSF tracer in Aqp4 KO mice We injected the fluorescent tracer Texas Red-dextran (3 kD, TRd3) intracisternally, as previously described (Iliff et al., 2012). We first compared overall penetration of TRd3 into the dorsal and ventral surfaces of the whole-brain between WT and Aqp4 KO (Aqp4-/-) mice using ex vivo near infrared fluorescence imaging. Quantification of the mean integrated optical density (MIOD) of TRd3 on the dorsal and ventral brain surface along anterior-posterior position of bregma showed a significant reduction in Aqp4 KO mice compared with WT mice (Figure 2a–c). Interestingly, CSF tracer entry into the ventral brain was higher than into the dorsal brain. We also compared penetration of TRd3 into the brain on the serial coronal forebrain slides (+1.7 to −0.7 mm from bregma) between the two genotypes. The percentage area of whole-slice fluorescence was significantly reduced in Aqp4 KO mice compared to WT controls (Figure 2d,f). Subregional quantification on the coronal section at the level of 0.5 mm anterior to the bregma showed that penetration of CSF tracer was high in the ventral and lateral brain surface of WT mice, but was comparable in the dorsal surface of the brain with Aqp4 KO mice (Figure 2g–h), further supporting difference in brain regions of CSF tracer influx. Specifically, Aqp4 KO markedly impaired the influx of TR-d3 into both the perivascular space and the brain parenchyma in the hypothalamus, one of the brain regions with the highest expression of AQP4 (Fan et al., 2005; Nielsen et al., 1997) (Figure 2e). Quantification of the intensity of TR-d3 as a function of the distance from the brain surface showed rapid decay of tracer with increasing distance from the ventral surface of the hypothalamus in Aqp4 KO mice. The tracer was almost undetectable at 500 the brain surface in both the perivascular space and in the brain parenchyma in Aqp4 KO mice. the TR-d3 fluorescence intensity at of the pial surface intensity to a depth of 500 in the perivascular space as well as in the adjacent parenchyma in WT mice (Figure replicate the finding that AQP4 the transfer of intracisternally injected TR-d3 from the CSF into the parenchyma (Iliff et al., 2012). RIKEN: of CSF tracer compromised tracer infiltration in Aqp4 KO mice The RIKEN and group used strain of Aqp4 KO mice, in which the exon 1 of Aqp4 is with In this experiment, biotinylated dextran was injected under ketamine-xylazine (Figure Because Aqp4 KO mice we used to This approach has an that BDA signals are by the and from fluorescence As a BDA distribution in the cortex of Aqp4 KO mice (Figure Quantification of SA-enhanced BDA signals in the cortical parenchyma showed profiles that with cortical depth after the for both and Aqp4 KO mice. BDA signals in the cortex of Aqp4 KO mice are of intensity and into the parenchyma at an anterior-posterior position of bregma (Figure we the compromised BDA penetration in Aqp4 KO mice is across the anterior-posterior of the cortex. Figure shows that BDA distribution in Aqp4 KO cortex is UNC: CSF tracer influx is decreased in Aqp4 KO mice compared to background strain controls and wild-type mice We the of the experiments performed in Smith et al. (2017) by using the same Aqp4 KO mouse line but the reported by Iliff et al. In to the background of the different Aqp4 KO lines have an effect on CSF we included both CD1 and C57BL/6 controls. We compared the entry of Fluor bovine serum into magna of mice. Thirty minutes after the injection, the brains were and tracer distribution evaluated in coronal sections as described previously (Iliff et al., 2012). distribution of tracer showed a significant reduction in Aqp4 KO mice compared with both CD1 mice (Aqp4+/+) and wild-type C57BL/6 (WT) mice (Figure significant difference in global tracer influx was seen between the two control groups. CSF influx was decreased or only to a brain we analyzed sections at different anterior-posterior as analyzed in Smith et al. (2017) (Figure in CSF influx between Aqp4 KO and controls was most in coronal sections anterior to bregma (Figure that sections the cortical of the cerebral a of CSF influx. quantification showed that this difference was the of decreased tracer penetration in the hypothalamus and of the basal forebrain (Figure Interestingly, CSF tracer entry along the ventral and lateral cortex was highest in the WT compared to the Aqp4+/+ control (Figure We that it is to replicate the presented in Iliff et al. in the mouse line reported by Smith et al. using URMC: cerebrospinal fluid entry to brain along the glymphatic pathway and is facilitated by the of AQP4 water the entry pathways of CSF to the brain we infused bovine serum and a Texas Red 3 kDa dextran into the magna of anesthetized WT and Aqp4 KO mice. In vivo transcranial optical imaging (Plog et al., of the BSA-647 showed CSF influx in the WT compared to the KO (Figure and Tracer the brain parenchyma through a of perivascular spaces of the cerebral on the pial tracer could be found on the dorsal cortical surface of WT mice compared to Aqp4 KO mice. After 30 brains were and The tracer distribution was quantified in coronal sections of both WT and Aqp4 mice. The KO mice influx for both the 3 kDa dextran and the 66 kDa BSA compared to the WT and despite the difference in molecular (Figure In a replicate experiment where the injection volume and injection rate was half of the Aqp4 deletion also reduced CSF entry through the glymphatic pathway by (Figure and The relative suppression of CSF tracer influx is comparable across previously reported despite use of different tracer and injection (Iliff et al., 2012). The depth of tracer penetration from the cortical surface was quantified using a approach to that reported in Smith et al. (2017) (Figure and Tracer penetration into brain was found to be higher in the WT than in the KO mice (Figure that the mice were in mice were using qPCR for both the Aqp4 locus (Figure and the excision sequence (Figure The that all mice were either for the Aqp4 locus (Aqp4+/+) or were (Aqp4-/-) of exon of the Aqp4 reduced glymphatic CSF influx in Snta1 KO mice with loss of expression of AQP4 in endfeet of the role of perivascular astrocytic localization of AQP4 in CSF into the brain we the Snta1 KO mouse mice lack expression of the protein which AQP4 to the and is critical for of the perivascular localization of AQP4 (Jessen et al., 2015). of AQP4 the loss of AQP4 perivascular localization in the Snta1 KO mice (Figure magnification imaging perivascular localization is AQP4 expression is by with a of the mouse line (Figure et al., We to CSF influx are perivascular AQP4 localization is we used dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to the influx of the contrast agent into brain parenchyma after intracisternal injection. We performed serial T1-weighted imaging at 10 min following of the contrast agent (Figure 30 min after the of the injection, levels of were in both WT mice and Snta1 KO mice, along the ventral surface of the brain (Figure but by 60 min signal within the parenchyma of Snta1 KO mice was significantly reduced compared to mice (Figure decreased signal in Snta1 KO mice across brain regions, which was most in cortex and hippocampus compared to subcortical brain regions and within the (Figure data a role for α-syntrophin in CSF-ISF exchange by support the reported role of AQP4 in this that the perivascular localization of AQP4 contributes to the of CSF influx into the brain parenchyma. of an injection in is linked to a global suppression of CSF influx Smith et al. (2017) questioned the existence of bulk flow within the brain parenchyma based on imaging tracer dispersion following injections in cortex or striatum. The tracers were by glass pipettes through a cranial The with insertion of glass pipettes is and traumatic injury markedly suppress CSF influx (Iliff et al., we questioned the of interstitial tracer dispersion using an invasive this we compared CSF tracer 66 influx in three groups of mice anesthetized with (i) controls with CM injection, (ii) insertion of a glass and injection of HiLyte 488-amyloid-β1-40 in or (iii) same as group two but HiLyte 488-amyloid-β1-40 was not to the aCSF (Figure One hour later, the BSA-647 tracer was injected into magna and to for 1 the brain was and the distribution of BSA-647 analyzed in coronal brain sections (Figure The analysis showed that the two groups to invasive markedly CSF tracer influx compared with the control group (Figure The