Young (2–4 months) and old (21–25 months) male wild type C57Bl/6 mice were obtained from the NIA colonies at The Jackson Laboratory. The mice were group-housed (12 h light/dark cycle) in a pathogen free environment. Mice were fed standard mouse chow (5,053 - PicoLab® Rodent Diet 20; 20% protein, 4.5% fat). All animal care and experimental protocols were approved by the University of California, San Francisco Institutional Animal Care and Use Committee.

Mice received one subcutaneous injection of monosodium glutamate (MSG) (3.5 g/kg) or isomolar sodium chloride solution as vehicle (0.02 M/kg or 1.2 g/kg). All solutions were prepared on the day of injection and filter sterilized. One day after injection, mice were perfused and the brains processed for histology.

Evans blue dye (1% w/v in sterile saline) was prepared on the day of injection and filtered sterilized. Mice were briefly anesthetized using an isoflurane chamber and then quickly administered 50 μL of Evans blue solution retro-orbitally. Perfusion with 4% (w/v) paraformaldehyde (PFA) occurred 20 min later. Direct fluorescence emitted by Evans blue was captured following frozen cryosectioning using an Olympus BX51WI microscope equipped with a QImaging Retiga 2000R digital camera. To measure EB permeability, EB permeable area instead of fluorescence intensity was measured since EB fluorescence intensity includes signals within the blood vessel lumens, intracellular as well as extracellular space, which does not reflect the permeable area. Measured values reflect the area of the ARC or AP with Evans blue dye.

Mice were anesthetized with an intraperitoneal injection of Avertin (250 mg/kg; Sigma Aldrich #T48402) followed by perfusion with 4% (w/v) PFA. Brains were post-fixed in 4% PFA, immersed in 30% sucrose overnight, and coronally sectioned at 20 μm on a cryostat. Immunofluorescent staining was performed using the following antibodies: rabbit anti-Vimentin (1:1000, Abcam #ab92547), rat anti-PVLAP (1:200, DHSB #MECA-32-S), rabbit anti-Collagen IV (1:1000, Abcam #ab6586), goat anti-Collagen IV (1:500, Novus Biologicals #NBP1-26549), and mouse anti-GFAP (1:1000, Cell Signaling Technology #3670). Sections underwent heat-mediated antigen retrieval in a 10 mM citrate solution. Following incubation with a blocking solution for 1 h, primary antibodies were added to sections and left overnight at 4 °C. Secondary antibodies were applied for 2 h in the dark at room temperature. Washes with PBS-T (0.1% Triton-X) occurred between each solution.

To detect Vegfa, Ptn, Vimentin, Plvap mRNA, RNAscope® fluorescent assay (Advanced Cell Diagnostics Inc., Hayward, CA) was employed. The Multiplex Fluorescent Reagent Kit v2 (ACD, Cat no. 323100) was used with the Opal 520 (Akoya Biosciences, Cat no. FP1487001KT, 1:1500), 620 (Akoya Biosciences, Cat no. FP1495001KT, 1:1500), and 690 (Akoya Biosciences, Cat no. FP1497001KT, 1:1500) Reagent Packs. The following probes from ACD were used Mm-Ptn-C3 (Cat no. 486381-C3), Mm-Vegfa-ver2 (Cat no. 412261), Mm-Vim-C3 (Cat no. 457961-C3), and Mm-Plvap-C2 (Cat no. 440221-C2). Sections (20 μm) were imaged on the Leica SP8 inverted laser scanning confocal.

Data analysis was performed using ImageJ/FIJI. The bregma of brain sections was defined using the Allen Brain Atlas and “The Mouse Brain- In Stereotaxic Coordinates” by Franklin and Paxinos. Images were blinded prior to quantification using A Better Finder Rename 11 software, which randomized the file order and renamed the files with numeric identifiers.

To obtain tanycyte cell counts, DAPI-positive nuclei along the 3rd ventricular wall were quantified in 63x single-plane images of the mediobasal hypothalamus. To quantify microvessel loops in the hypothalamus, PLVAP-expressing vessels extending from the outer wall of the ME were visualized from Bregma −1.7 to −2.1 to obtain the average count per section. To measure glial and microvessel loop interactions, stained sections were imaged on the Leica SP8 inverted laser scanning confocal microscope. Using binarized single confocal plane images, the area of overlapping pixels between channels for glial cells and blood vessels was defined as an “interaction.” Data were expressed as a percentage of capillary surface coverage, based on Collagen IV area. Interactions were measured along 200 μm of the ME capillary bed for each image. To measure fenestrated capillaries in the AP, a binary threshold was performed on fluorescent images to segment signal-positive regions. Total fluorescent signal area was then quantified as a readout for capillary abundance. Vessels in the AP were visualized from Bregma −7.56 to −7.64.

To quantify mRNA expression in hypothalamic tanycytes, which are densely juxtaposed, fluorescent intensity was measured as a proxy for transcript abundance. To achieve tanycyte subtype specific expression, the DAPI-labeled nuclei of the 3rd ventricle were divided into subtype regions based on morphology and localization. On the floor of the 3 V, β2-tanycytes were defined by the vertical polarity of their cell bodies and extension of their processes onto the ME capillary bed. The border between the β2- and β1-tanycytes was defined where the orientation of the polarity of the cell bodies changed from vertical to angled. The cells lining the ventrolateral portion of the 3 V were considered β1-tanycytes, who project their curved processes at the ME/ARC border. Dorsal to β1-tanycytes, α2-tanycytes were defined as the cells lining the lateral 3 V adjacent to the ARC and ventromedial hypothalamus. In the AP, where tanycyte-like cells are more dispersed, fluorescent signal was binarized, and the area of positive signal represented mRNA expression levels.

An unpaired two-tailed student’s t-test was employed to compare two independent groups. For comparisons involving two genotypes and multiple treatments or conditions, an ordinary or repeated-measures two-way ANOVA with multiple comparisons was utilized. Normality of the data was analyzed using the Shapiro–Wilk test. In the event that a dataset did not pass the normality test, a nonparametric test was used. All statistical analyses were conducted using GraphPad Prism 10.5.0 software (GraphPad Software, Inc., La Jolla, CA, USA).

We have previously shown that hypothalamic neurons outside the BBB, but not those that are protected by the BBB, are rapidly ablated by peripheral administration of MSG, a glutamate that does not cross the BBB and ablates neurons by excitotoxicity (Yulyaningsih et al., 2017). We sought to determine if fenestrated vascular permeability in the ME was modulated upon exposure to MSG, and if aging affected such response. To this end, young (2–4 months) and old (21–25 months) male mice were injected subcutaneously with MSG or vehicle (isomolar sodium chloride). One day later, mice were injected with Evans blue, a BBB-impermeable fluorescent dye, right before perfusion. Consistent with what we reported previously (Yulyaningsih et al., 2017; Olofsson et al., 2013), cells in the ME and mediobasal ARC were positive for Evans blue (Figure 1A). Intriguingly, the area of Evans blue permeability in the ARC parenchyma significantly decreased 1 day after MSG treatment in young adult mice but not in aged mice (Figures 1A,C).

Since fenestrated capillaries permit the permeation of Evans blue into the ME and ARC, we visualized fenestrated capillaries by immunostaining of plasmalemma vesicle-associated protein (PLVAP), the protein that makes up the sieve-like diaphragm of the fenestrae in fenestrated endothelial cells and hence specifically marks fenestrated blood vessels (Stan et al., 2012; Denzer et al., 2023). All blood vessels, both fenestrated and non-fenestrated, were visualized by immunostaining with Collagen IV, a component of the basement membrane of blood vessels. PLVAP-positive capillaries were located at the outer edge of the ME with microvessal loops extending into the ME and ARC parenchyma (Figure 1B). Numbers of these PLVAP⁺ fenestrated microvessal loops were reduced 1 day after MSG treatment in young adults but not in aged male mice (Figures 1B,D). Together, these results suggest that the abundance of fenestrated microvessel loops and consequently fenestrated vascular permeability at the ME and ARC decrease upon MSG exposure. Importantly, this regulation is abolished in aged animals, suggesting that aging impairs the ability of fenestrated blood vessels to remodel in response to circulating neurotoxins.

Glial cells play essential roles in regulating vascular function in the brain. In most brain regions, astrocyte endfeet form a complex network surrounding barrier endothelial cells and this close interaction is vital for the integrity and functionality of the BBB (Abbott et al., 2010). The hypothalamus also contains tanycytes, which are specialized radial glia-like cells that line the wall of the 3^rd ventricle. Tanycyte cell bodies are located on the ventricular wall, and each tanycyte extends a single long basal process into the ME or hypothalamic parenchyma (Rodriguez et al., 2005; Langlet et al., 2013). As glial processes may serve as a physical barrier to restrict the diffusion of blood-borne substances from the ME into the ARC parenchyma, we examined the physical interaction between astrocyte or tanycyte endfeet and fenestrated capillaries. Hypothalamic sections from young adult male mice were immunostained with a PLVAP antibody to mark fenestrated capillaries, a Collagen IV antibody to visualize the basement membrane of all blood vessels, a GFAP antibody to label astrocytes, and a Vimentin antibody to reveal tanycytes (Figures 2A,B). Single-plane confocal images were processed and the area of contact between endfeet of astrocytes or tanycytes and fenestrated capillaries in the ME perimeter was quantified (Supplementary Figure 1). Fenestrated capillaries exhibited a strikingly high degree of physical contact with tanycytes, and to a much lesser extent with astrocytes (Figures 2C,D; Supplementary Figure 1). The high degree of physical contact between tanycyte endfeet and fenestrated capillaries suggests that tanycytes may exert significant influence on fenestrated endothelial cells.

Astrocytic contact with fenestrated capillaries increased with age, while tanycytic contact did not change. There was also no change in astrocytic contact or tanycytic contact with fenestrated capillaries in young mice in response to MSG treatment (Figures 2C,D; Supplementary Figure 2). Neither MSG treatment or age affected β1, β2 tanycyte numbers (Figures 2E,F; Supplementary Figure 2), or β1 tanycyte processes which may act as a physical barrier between ME and ARC (Figure 2G). Thus, these data do not support the notion that MSG-induced reduction in fenestrated vascular permeability in young mice is attributed to gross structural changes of tanycytes or astrocytes.

Since tanycytes show close contact with fenestrated capillaries, they may regulate fenestrated vasculature remodeling through secreted angiogenic factors. We examined a list of tanycyte-enriched genes from published single-cell RNA-sequencing data from the adult hypothalamus (Chen et al., 2017). Vegfa was one of such genes (#41 on the list) [Table S4 in Chen et al. (2017)]. Notably, Ptn was identified as a top tanycyte-enriched gene (#2 on the list) [Table S4 in Chen et al. (2017)]. PTN, also named heparin affinity regulatory peptide (HARP) in some studies, is a potent angiogenic factor, which stimulates proliferation and migration of endothelial cells during normal development and in cancer (Zhang and Deuel, 1999; Perez-Pinera et al., 2007; Perez-Pinera et al., 2008; Papadimitriou et al., 2022; Papadimitriou et al., 2009; Mikelis and Papadimitriou, 2008; Mikelis et al., 2007). In vitro studies show that PTN acts on endothelial cells to stimulate their migration and promote their formation of tube-like structures (Papadimitriou et al., 2001; Souttou et al., 2001). In vivo, PTN alone is sufficient to initiate new blood vessel formation that is both normal in appearance and function (Christman et al., 2005). PTN expression is high in the developing brain, peaks at birth but declines as the brain matures (Kadomatsu and Muramatsu, 2004). Despite this, expression and function of PTN in the adult brain are not well characterized.

We next examined Ptn and Vegfa mRNA expression in tanycytes of the adult hypothalamus of male mice by RNAscope. Tanycyte cell bodies line the 3^rd ventricular wall and are classified into 4 different subtypes based on their locations: α1 tanycytes line the most dorsal wall of the 3^rd ventricle, whereas α2 tanycytes occupy the ventral ventricular wall adjacent to the ARC; β1, β2 tanycytes line the lateral evaginations and floor of the 3^rd ventricle, respectively. Ptn mRNA expression was abundant but restricted to α2, β1 and β2 tanycytes of adult mice, whereas Vegfa expression was most highly expressed in β2 tanycytes (Figure 3A). Ptn expressions in β1 and β2 tanycytes, cells that directly contact fenestrated capillaries with their endfeet, were reduced in young adult mice after MSG-induced lesioning, and such regulation was abolished in the aged mice (Figure 3C). In contrast, Ptn expression in α2 tanycytes, which project into the hypothalamic parenchyma, was not altered by MSG treatment in either young or aged mice (Figure 3C). Vegfa expression in β2 tanycytes also followed a similar trend (Figure 3D). Notably, tanycytic Ptn expression significantly correlated with Vegfa expression (Figure 3E) and with Evans blue permeability in the ME and mediobasal ARC (Figure 3F). Together, these results suggest that expressions of angiogenic factors VEGF and PTN are modulated in response to MSG. As the basal processes of β1 and β2 tanycytes directly contact the fenestrated capillaries, our results suggest that tanycytic release of angiogenic factors VEGF and PTN may regulate the remodeling of fenestrated blood vessels in response to circulating neurotoxins. Our finding also suggests that aging impairs the ability of tanycytes to modulate the expression of angiogenic factors, which may underlie the loss of fenestrated capillary plasticity in aging.

To evaluate if MSG-induced remodeling of fenestrated vasculature is unique to the ME, we examined the AP, another circumventricular organ in the brainstem in male mice. Surprisingly, the Evans blue permeable region in the AP of young adult mice was not reduced but rather increased in response to MSG treatment and again this modulation was significantly diminished in the aged mice (Figures 4A,J). Consistently, the abundance of PLVAP-positive fenestrated capillaries in the AP were increased upon MSG treatment in young adults but not in aged mice (Figures 4B,K). Vimentin-positive tanycyte-like cells were present in the AP (Figures 4C–E), but unlike the β tanycytes at the ME which do not express GFAP, tanycyte-like cells in the AP co-expressed Vimentin and GFAP (Figure 4C). Notably, mRNA expression of Vegfa and Ptn were highly enriched in the AP (Figure 4F). Vegfa and Ptn were not expressed by Plvap⁺ endothelial cells (Figure 4G). Only about 40% of Vegfa-expressing cells in the AP expressed Vimentin (Figure 4H). However, near 80% Ptn-expressing cells co-expressed Vimentin, suggesting that PTN is mainly produced by tanycyte-like cells in the AP (Figure 4I). Notably, both Vegfa and Ptn expression were increased upon MSG treatment, and this regulation was abolished in aged mice (Figures 4L,M). Together, these results suggest that fenestrated capillaries in the AP undergo distinct remodeling compared with those in the ME upon exposure to circulating neurotoxins and that this regulation is impaired in aging. The concordant regulation of Vegfa and Ptn expression suggests that modulation of angiogenic factors in the AP may play a key role in the remodeling of fenestrated vasculature in this region and that this regulation is lost in aged animals.