Decision letter: Apolipoprotein M-bound sphingosine-1-phosphate regulates blood–brain barrier paracellular permeability and transcytosis

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 blood-brain barrier (BBB) is formed by the endothelial cells lining cerebral microvessels, but how blood-borne signaling molecules influence permeability is incompletely understood. We here examined how the apolipoprotein M (apoM)-bound sphingosine 1–phosphate (S1P) signaling pathway affects the BBB in different categories of cerebral microvessels using ApoM deficient mice (Apom-/-). We used two-photon microscopy to monitor BBB permeability of sodium fluorescein (376 Da), Alexa Fluor (643 Da), and fluorescent albumin (45 kDA). We show that BBB permeability to small molecules increases in Apom-/- mice. Vesicle-mediated transfer of albumin in arterioles increased 3 to 10-fold in Apom-/- mice, whereas transcytosis in capillaries and venules remained unchanged. The S1P receptor 1 agonist SEW2871 rapidly normalized paracellular BBB permeability in Apom-/- mice, and inhibited transcytosis in penetrating arterioles, but not in pial arterioles. Thus, apoM-bound S1P maintains low paracellular BBB permeability in all cerebral microvessels and low levels of vesicle-mediated transport in penetrating arterioles. Introduction The blood-brain barrier (BBB) allows only certain molecules and cells to pass from the blood circulation to the brain. Molecules cross the BBB via the paracellular route by diffusion between endothelial cells (Tietz and Engelhardt, 2015), or by transcellular route, including vesicular transport across the endothelium (De Bock et al., 2016). The selectivity of the BBB towards small molecules is maintained by tight and adherens junctions between adjoining endothelial cells (Tietz and Engelhardt, 2015), whereas the transport of large molecules is restricted by receptor-mediated interactions (De Bock et al., 2016). Under normal conditions, the BBB maintains low permeability due to the structural barrier maintained by junctional complexes and the low rate of transcytosis (De Bock et al., 2016; Daneman and Prat, 2015; Nico and Ribatti, 2012; Abbott et al., 2010). In disease states, the loss of BBB function leads to increased permeability, which is viewed mainly as the consequence of disrupted junctional complexes at endothelial cell contact sites (Davies, 2002; Engelhardt et al., 2014; Salameh et al., 2016). However, recent evidence suggests equal importance of transcytosis, as a failure in the homeostatic regulation of endothelial trans-cellular transport may aggravate brain pathologies (Knowland et al., 2014; Habgood et al., 2007; Hashizume and Black, 2002; Krueger et al., 2013). Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid synthesized by all mammalian cells, with blood-circulating S1P produced by vascular endothelial cells, platelets, and erythrocytes (Thuy et al., 2014; Blaho and Hla, 2014; Xiong and Hla, 2014; Prager et al., 2015). S1P is a ligand for five membrane-bound G-protein-coupled receptors (S1PR1-5), and the brain endothelium expresses S1PR1-3 (Prager et al., 2015). The majority (~70%) of S1P in the blood circulation is transported by apolipoprotein M (apoM), a 26 kDa protein mainly associated with high-density lipoprotein (HDL) (Xu and Dahlbäck, 1999), while the remaining S1P fraction is carried primarily by albumin (Ruiz et al., 2017; Christoffersen et al., 2011). Depending on the carrier (i.e., apoM or albumin), S1P preferentially activates S1PR1 or S1PR2 and S1PR3, which can exert opposite effects (Murata et al., 2000). For example, S1PR1 stimulation inhibits neuroinflammation, whereas S1PR2 and S1PR3 stimulation facilitates pro-inflammatory signaling cascades (Sanchez et al., 2007; Sanchez, 2016; Blaho et al., 2015). The onset and severity of sepsis in humans correlate with reduced apoM-S1P plasma concentrations and loss of BBB integrity (Davies, 2002; Kumaraswamy et al., 2012). In mice, lack of apoM compromises the endothelial barrier in the lungs and brown adipose tissue (Christoffersen et al., 2011; Christensen et al., 2016; Christoffersen et al., 2018), but its effects on the BBB are largely unexplored. First, apoM-deficient mice are less protected against neuroinflammation (Blaho et al., 2015). Second, conditional knock-out of S1PR1 increases the BBB permeability towards small molecules (Yanagida et al., 2017), but no data are available on how the BBB properties change with consistent S1PR1 expression, but during reduced apoM levels. Our objective was to determine the modulatory role of apoM for the BBB. We used two-photon microscopy to image the brains of apoM-deficient (Apom-/-) mice in vivo in order to characterize changes in BBB permeability in different categories of cerebral microvessels. Lack of apoM increased the BBB permeability to small molecules without affecting the ultrastructural components of junctional complexes and caused substantial increases in adsorptive transcytosis in pial and penetrating arterioles, but not in venules and capillaries. Systemic administration of a selective S1PR1 agonist SEW2871 promptly reversed the increased permeability for the paracellular route, whereas the normal rate of transcytosis was restored only in penetrating arterioles. We suggest that modulation of the apoM signaling axis may be clinically relevant, but heterogeneous responses of distinct vessel types to apoM shortage and S1PR1 agonist treatment should be taken into account in the development of protective strategies in neurological conditions. Results Deficiency in apoM increases BBB permeability To assess the BBB with all structural constituents we used two-photon fluorescence imaging in vivo in transgenic apoM-deficient mice (Apom-/-) mice (Christoffersen et al., 2011), which in contrast to other animal models, e.g. S1pr1iECKO (S1pr1 knock-out) mice (Yanagida et al., 2017), retain expression and function of S1PR1 (Christoffersen et al., 2011). Following preparative surgery (Figure 1a), the brain was imaged in living anesthetized animals through an acute craniotomy over the somatosensory cortex (Figure 1b). Figure 1 with 1 supplement see all Download asset Open asset Two-photon imaging of paracellular permeability changes in Apom-/- mice. (a) Schematic of a mouse after preparative microsurgery for two-photon imaging, with (b) craniotomy over the somatosensory cortex. (c) Three-dimensional reconstruction of the brain microvasculature from a single Z-stack after i.v. injection of NaFluo. Each Z-stack corresponds to a single time point and underwent dimensionality reduction by maximum intensity projection along the Z-axis (depth). (d) Relative fluorescence change over the baseline (first projected Z-stack in the recording). Contours delineate the main cerebral microvasculature. Compared to WT, the Apom-/- animals had increased accumulation of NaFluo in the brain parenchyma over time, which was ameliorated by treatment with S1PR1 agonist SEW2871. (e) Bolus injections of fluorophore were separated by 30 min time-lapse imaging intervals. The panels show fluorescent molecule accumulation in the brain parenchyma for all tested fluorophores at 30 min post-injection. (f) Changes in fluorescence intensity over time in the brain parenchyma and the blood circulation expressed as a fraction of the baseline. (g) Quantitative assessment of the total effect 30 min post-injection by area under the curve (AUC) calculation. Left bar plot: increased paracellular permeability of the BBB in Apom-/- mice towards sodium fluorescein (0.365 kDa) and Alexa Fluor488 (0.643 kDa), but not large molecule FITC-dextran (10 kDa). Right bar plot: no differences in the signal decrease of blood-circulating fluorophores among all experimental groups. (h) The mean arterial blood pressure during imaging was the same in all experimental groups. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. First, the integrity of the BBB was assessed after an i.v. bolus injection of sodium fluorescein (NaFluo). NaFluo is a small (0.365 kDa), negatively charged hydrophilic molecule, which determines its preferred route across the BBB via paracellular diffusion, and has been shown to be suitable for characterizing the paracellular leak, independently from vesicular transport across the barrier (Cheng et al., 2010; Hawkins and Egleton, 2006; Kozler and Pokorný, 2003). The fluorescence signal from blood-circulating fluorophore and brain parenchyma was simultaneously measured for 30 min in a series of Z-stacks of relatively large brain volume (750 μm x 750 μm x 114 μm) that contained pial and penetrating microvessels, and capillaries (Figure 1c). Each Z-stack underwent dimensionality reduction, that is, maximum intensity projection along the Z (depth) axis. The changes in fluorescence in brain parenchyma were not apparent on raw fluorescence data images (Figure 1c), therefore we chose to represent the data as relative increases of fluorescence over the baseline (Figure 1d–e). The morphology of the vasculature delineated by blood-circulating NaFluo did not differ between WT and Apom-/- mice (Figure 1d). We detected a progressive accumulation of NaFluo in the brain parenchyma in both WT and apoM-deficient mice. However, Apom-/- mice exhibited considerably higher levels of fluorophore accumulation in the brain compared to WT mice already at 15 min after injection, with a clear difference at the 30 min time-point (Figure 1d, Video 1). As apoM is the main carrier for S1P in the blood stream and S1P bound to apoM primarily activates S1PR1 on the endothelial cells (Prager et al., 2015; Murata et al., 2000), we tested whether the increase in BBB permeability due to the lack of apoM was a consequence of the S1PR1 hypostimulation. The Apom-/- mice were i.p. injected 150 min prior to imaging with a selective S1PR1 agonist, SEW2871. Compared to untreated Apom-/-, the SEW2871-treated Apom-/- mice exhibited decreased permeation of NaFluo into the brain (Figure 1d, Video 1), suggesting that the S1PR1 agonist rescued the abnormal Apom-/- BBB phenotype. This result indicates that the apoM deficiency impairs S1PR1 signaling to the degree that causes increased BBB permeability. We next examined whether the BBB also exhibited increased permeability towards other molecules of different sizes and chemical properties. Each animal in the WT, Apom-/-, and Apom-/- SEW2871-treated group was injected at 30 min intervals with fluorophores of decreasing molecular size, starting with 10 kDa FITC-dextran, followed by 0.643 kDa Alexa Fluor 488 and 0.365 kDa NaFluo (Figure 1e). After each injection, we imaged the same brain volume over time, with subsequent dimensionality reduction of the Z-stacks. To compare animal groups, we measured the changes in fluorescence signal over time from ROIs placed in the brain parenchyma and vasculature (n = 7 mice in each group), and accounted for systematic error in measurement of consecutively injected fluorophores (see Methods section, Figure 1—figure supplement 1). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Lack of apoM leads to a reversible increase in BBB paracellular permeability. Accumulation of NaFluo in brain parenchyma presented as fluorescence increase relative to the baseline. Fluorophore injections occurred 1 min before the first recorded imaging plane. For both tracer molecules, Alexa Fluor 488 and NaFluo, and in all animal groups, we detected fluorescence increase over time in the parenchyma, and the effect was persistent without the signal reaching saturation (Figure 1f). In contrast, there was no increase in brain fluorescence for FITC-dextran (Figure 1e–f). To better characterize the fluorescence increase over time, we calculated the area under the curve (AUC) of the signal traces, that is, the cumulative fluorescence increase for the duration of 30 min after each fluorophore injection (Figure 1f–g). We observed no differences in BBB permeability between WT and Apom-/- mice for 10 kDa FITC-dextran (WT 0.0086 ± 0.0056 min vs. Apom-/- 0.033 ± 0.023 min, p=0.3068), but the BBB in Apom-/- mice was significantly more permeable to small molecules, such as Alexa Fluor 488 (WT 0.90 ± 0.31 min vs. Apom-/- 2.43 ± 0.61 min, p<0.05) and NaFluo (WT 5.11 ± 1.3 min vs. Apom-/- 27.5 ± 4.9 min, p<0.001) (Figure 1g, Videos 2–3). Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg WT mice BBB paracellular permeability towards NaFluo, Alexa Fluor 488, and 10 kDa FITC-dx. Accumulation of fluorophores in brain parenchyma presented as fluorescence increase relative to the baseline. For each time-lapse recording, the respective fluorophore was injected 1 min before the first recorded imaging plane. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Increase in the BBB paracellular permeability in Apom-/- mice towards NaFluo and Alexa Fluor 488, but not 10 kDa FITC-dx. Accumulation of fluorophores in brain parenchyma presented as fluorescence increase relative to the baseline. For each time-lapse recording, the respective fluorophore was injected 1 min before the first recorded imaging plane. Treatment of the Apom-/- mice with SEW2871 markedly reduced the leakage of both Alexa Fluor 488 and NaFluo compared to untreated Apom-/- mice (0.75 ± 0.38 vs. 2.43 ± 0.61 min, p<0.05; and 5.45 ± 1.6 min vs. 27.5 ± 4.9 min, p<0.01; Figure 1e–g, Videos 3–4). The reversal of the dysfunctional BBB phenotype occurred relatively fast, and at 150 min post-treatment with SEW2871, the BBB permeability was the same in Apom-/- and WT mice (p=0.6949 and p=0.8778 for Alexa Fluor 488 and NaFluo, respectively; Figure 1g). In addition we performed the same analysis on arbitrary fluorescence units [a.u.], that is non-normalized data (Figure 1—figure supplement 1e–f) and the results were in accord with relative fluorescence increases. We chose to report the data as relative changes for the results to be more easily comparable between different imaging setups. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Apom-/- SEW2781-treated mice exhibit normal BBB paracellular permeability towards NaFluo, Alexa Fluor 488, and 10 kDa FITC-dx. Accumulation of fluorophores in brain parenchyma presented as fluorescence increase relative to the baseline. For each time-lapse recording, the respective fluorophore was injected 1 min before the first recorded imaging plane. Importantly, the decrease in fluorescence signal in vessels (i.e., clearance of the respective fluorophore from the blood circulation) did not differ between WT, Apom-/-and Apom-/- SEW2871-treated mice for each respective fluorophore (Figure 1f–g). In addition, we found no differences in mean arterial blood pressure between animal groups before (pre-imaging) or after the administration of all fluorophores (post-imaging) (Figure 1h). Thus, the increased fluorescence accumulation in the brains of Apom-/- mice and reduced accumulation of fluorophores in the brains of SEW2871-treated Apom-/- mice were not caused by different kinetics of a fluorophore clearance from the blood stream or differences in blood pressure. These results show that apoM shortage increases the BBB permeability towards small molecules (~0.3–0.7 kDa), and that the effect can be reversed by S1PR1 stimulation. Deficit in apoM signaling does not alter BBB tight junction ultrastructure Increases in the BBB permeability towards small molecules may be indicative of defective structural elements that restrict paracellular diffusion across the BBB, e.g. junctional complexes (Davies, 2002; Engelhardt et al., 2014; Salameh et al., 2016). Therefore, we next assessed the ultrastructure of the BBB using transmission electron microscopy (TEM). We analyzed brain microvessels (<6 µm) cross-sections from WT, Apom-/-, and SEW2871-treated Apom-/- mice. In contrast to capillaries, large vessels were susceptible to perfusion-fixation artifacts (non-uniform distortion of endothelium neighboring large tissue-devoid areas, i.e. vessel lumen), which rendered pial and penetrating vessels not suitable for TEM quantitative assessment of the BBB ultrastructure. In each vessel, we measured the thickness of the endothelium, basement membrane (both n = 104 vessels; four mice in each group), tight junctional complexes, and the endothelial cleft, the percentage coverage of endothelial cell contact sites by junctional complexes (all n = 164, four mice in each group), and the endothelial cell area (n = 104 vessels, four mice in each group) (Figure 2, Figure 2—figure supplement 1). The width, length, and coverage of junctional complexes were the same for all three groups of mice (Figure 2a–b, Figure 2—figure supplement 1a), suggesting that the compromised BBB was not caused by a structural defect of junctions, and that the rescue effect of SEW2871 was not mediated by alteration of the morphology of these structural components. However, we detected an ~ 10% increase in the thickness of the endothelium in S1P-signaling-deficient mice compared to controls (Apom-/- 0.078±0.0026 μm vs. WT 0.071 ± 0.0025 µm; p<0.05) and the rescue group (Apom-/-+SEW2871 0.070 ± 0.0025 μm; p<0.05; Figure 2c). Notably, the stimulation of S1PR1 in Apom-/- mice decreased the endothelial cell area (5.80 ± 0.37 vs. 4.24 ± 0.23 μm2; p<0.001), with a strong trend for the cell area in Apom-/- to be larger than in WT mice (5.80 ± 0.37 vs. 4.78 ± 0.24 μm2; p=0.068; Figure 2d). These changes were relatively small, and they are unlikely to disrupt BBB structural integrity to induce robust permeability increase for small molecules. Altogether, our results suggest that the increased permeability observed in Apom-/- mice is not mediated by ultrastructural changes at the level of the junctional complexes. Figure 2 with 1 supplement see all Download asset Open asset Small changes in endothelium cell morphology, but not junctional complexes, accompany increased BBB paracellular permeability in Apom-/-mice. TEM ultrastructure assessment of the structural components of the BBB revealed no changes in the (a) width or (b) length of junctional complexes, with (c) a small increase in endothelial thickness in Apom-/- mice compared to WT mice and a decrease in SEW2871-treated Apom-/- mice. (d) A small decrease in endothelial cell area was observed upon SEW2871 treatment in Apom-/- mice. Data are presented as interquartile distributions with median (horizontal line) and mean (plus sign). The box whiskers indicate 10–90% percentiles. *p<0.05, ***p<0.001. ApoM/S1PR1 signaling deficit increases transcellular transport Next, we examined whether lack of apoM leads to increased transcytosis in the BBB. The animals were injected with bovine serum albumin conjugated to Alexa 488 (BSA-Alexa 488) into the blood stream to monitor vesicular transport across the endothelium (Knowland et al., 2014). Following the injections, the animals were continuously imaged for 120 min in a hyperstack mode with subsequent dimensionality reduction (as in Figure 1c). In all animal groups, and as early as 30 min post-injection, we detected BSA-Alexa 488-containing vesicles at the BBB interface (Figure 3). Compared to WT animals, Apom-/- mice exhibited increased albumin in endothelial cells, indicating that vesicular transport was increased in the absence of apoM. Furthermore, the treatment of Apom-/- animals with SEW2871 reversed the observed effect, suggesting that the same apoM-dependent mechanism underlies the previously observed increase in permeability for small fluorescent tracers, as well as elevated transcytosis. Figure 3 with 2 supplements see all Download asset Open asset Increase in BSA-Alexa 488 uptake indicates an increase in transcellular transport in Apom-/- mice. Two-photon assessment of albumin uptake (green puncta) 60 and 120 min post i.v. injection of BSA-Alexa 488. Arrowheads indicate numerous puncta of BSA-Alexa 488 vesicles at the BBB interface in Apom-/- mice but only sparse labeling in WT and SEW2871-treated animals. pa = pial arteriole; pe = penetrating arteriole; cap = capillaries; pv = pial venule; av = ascending venule. We detected no signs of fibrinogen entry into the brain parenchyma; and similarly to S1PR1 conditional KO mice (Yanagida et al., 2017), there were no differences in morphology of microglia/macrophages and astrocytes between WT and Apom-/- mice, suggesting lack of gliosis upon exposure to albumin (Figure 3—figure supplement 1). However, we detected in the brain parenchyma the presence of cells that sequestered blood-borne albumin (Figure 3). To identify the cells, we performed a series of immunohistochemical analysis on PFA-fixated brains of Apom-/- mice to investigate for the colocalization of cell-internalized BSA-Alexa488 with the immunofluorescence signal from ionized calcium binding adaptor molecule 1 (Iba-1, microglia/macrophages marker); transmembrane protein 119 (TMEM119, microglia-specific marker); RM0029-11H3 (monoclonal antibody against macrophage-specific epitope) and glial fibrillary acidic protein (GFAP, astroglia marker). First, we ascertained that the fixation protocol did not quench the signal from BSA-Alexa488 (Figure 3—figure supplement 2a). The albumin sequestering cells were to be on the colocalization of BSA-Alexa488 cells with and with antibody RM0029-11H3 signal (Figure 3—figure supplement was no colocalization of the BSA-Alexa488 signal with the microglia-specific and astroglia (Figure 3—figure supplement 2c). We found only signs puncta) of BSA-Alexa488 uptake by other cell types (Figure 3—figure supplement S1P signaling deficit transcytosis only in arterioles Next, we assessed whether the increase in transcellular transport caused by the deficiency was the same for all vessel The of vesicles was over time for each vessel pial arterioles, penetrating arterioles, capillaries, ascending and pial venules (n = The data are expressed as the expressed as the of vesicles of vessel area (Figure In all experimental groups, the of BSA-Alexa vesicles was on vessel and 120 min post-injection, the in WT mice was observed in capillaries and decreased with vessel of the vessel (i.e., arterioles or (Figure In Apom-/- mice, the deficiency in S1PR1 signaling increased transcytosis in pial and penetrating arterioles, but not in venules and capillaries. The effect was in penetrating arterioles, the vascular in Apom-/- mice was 10-fold higher than in WT mice (Apom-/- ± 0.071 vs. WT ± μm2; and three higher than for pial arterioles (Apom-/- vs. WT ± μm2; p<0.05; Figure The level of transcytosis did not change in Apom-/- mice in the capillaries, or pial and Figure 4 Download asset Open asset of different categories of brain microvessels to increased transcytosis in Apom-/- mice. (a) the of the of vessels against respective vessel 120 min post BSA-Alexa 488 The represent the into distinct vessel Data from capillaries were to the order of vessel change in the brain (b) Quantitative analysis the increase in albumin uptake in Apom-/- mice that was only in the arterial of the vascular and in capillaries and penetrating arterioles to SEW2871 in are the of vessels across five mice in each The data are presented as mean ± SEM. (c) the kinetics of the changes for WT, Apom-/-, and Apom-/- SEW2871 mice with to different vessel The data are presented as mean ± SEM. *p<0.05, but not pial arterioles to treatment S1PR1 stimulation with SEW2871 to of the transcellular transport of albumin in the penetrating arterioles of Apom-/- mice to the level of WT controls (Apom-/-+SEW2871 ± vs. WT ± Figure In contrast, SEW2871 treatment did not decrease transcytosis in pial arterioles (Apom-/-+SEW2871 ± vs. Apom-/- μm2; and albumin transport was still significantly elevated compared to WT mice (Apom-/-+SEW2871 ± vs. WT ± μm2; we assessed the kinetics of the vesicular increase over time with to each animal group and vessel In all vessel types and animal experimental groups, the increase in BSA-Alexa 488 was persistent and did not saturation at 120 min, indicating that the results were not by the transcellular transport with BSA-Alexa 488 (Figure These show that transcellular transport is in apoM-deficient mice, and that there is not only in the vasculature towards increased permeability, but also towards which vessels can be rescued by stimulation of the S1PR1 signaling Discussion the influence of blood-borne signaling molecules on brain endothelial cells is for an of the BBB We report that lack of apoM increased the BBB permeability for small molecules without changes in size, or of tight junction complexes. Furthermore, we report an to increase in adsorptive transcytosis in the endothelial cells of pial and penetrating arterioles, but not in venules and capillaries. Thus, ApoM the of small and large molecules to different in different vascular the increased BBB permeability for small molecules and large be rescued by selective stimulation of the the two-photon of the BBB in vivo are The presented data is first to show the of the BBB responses to S1PR1 and treatment in different vessel the of the BBB in a living brain with to different and of the This may be of importance in as S1PR1 modulation is used in in humans et al., 2010). among the used in BBB paracellular permeability we chose three molecules with different NaFluo and Alexa Fluor 488 that via the BBB, and FITC-dextran that has low BBB permeability et al., 2014; et al., The loss of BBB integrity in Apom-/- mice was observed for small tracer molecules but no was observed for 10 kDa which is in with in mice (Yanagida et al., However, in contrast to and (Yanagida et al., 2017), we show that is not the absence of brain endothelial S1PR1 but a lack of a fraction of S1P in the blood (i.e., apoM-bound is to the BBB, and the presence of S1PR1 (Christoffersen et al., 2011). we cannot that NaFluo molecules were in transport vesicles during at the of the endothelium, in our we did not vesicular fraction of NaFluo, Alexa Fluor 488 and at the BBB. In contrast, Alexa 488 clear vesicular which is consistent with transcytosis (De Bock et al., 2016; et al., et al., Furthermore, NaFluo is a strong for endothelium that is that the tracer to the blood stream et al., Therefore, we that the transcytosis of NaFluo, Alexa Fluor 488 or had to fluorescence and the effect observed in the brain parenchyma occurred due to the increase in paracellular permeability. The increased permeability along the paracellular pathway in response to and et al., et al., and is to be to the ultrastructural of junction