Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
BMC Biol
2025 Feb 11;231:43. doi: 10.1186/s12915-025-02145-7.
Show Gene links
Show Anatomy links
Species-specific blood-brain barrier permeability in amphibians.
Antesberger S
,
Stiening B
,
Forsthofer M
,
Joven Araus A
,
Eroglu E
,
Huber J
,
Heß M
,
Straka H
,
Sanchez-Gonzalez R
.
???displayArticle.abstract???
BACKGROUND: The blood-brain barrier (BBB) is a semipermeable interface that prevents the non-selective transport into the central nervous system. It controls the delivery of macromolecules fueling the brain metabolism and the immunological surveillance. The BBB permeability is locally regulated depending on the physiological requirements, maintaining the tissue homeostasis and influencing pathological conditions. Given its relevance in vertebrate CNS, it is surprising that little is known about the BBB in Amphibians, some of which are capable of adult CNS regeneration.
RESULTS: The BBB size threshold of the anuran Xenopus laevis (African clawed toad), as well as two urodele species, Ambystoma mexicanum (axolotl) and Pleurodeles waltl (Iberian ribbed newt), was evaluated under physiological conditions through the use of synthetic tracers. We detected important differences between the analyzed species. Xenopus exhibited a BBB with characteristics more similar to those observed in mammals, whereas the BBB of axolotl was found to be permeable to the 1 kDa tracer. The permeability of the 1 kDa tracer measured in Pleurodeles showed values in between axolotl and Xenopus vesseks. We confirmed that these differences are species-specific and not related to metamorphosis. In line with these results, the tight junction protein Claudin-5 was absent in axolotl, intermediate in Pleurodeles and showed full-coverage in Xenopus vessels. Interestingly, electron microscopy analysis and the retention pattern of the larger tracers (3 and 70 kDa) demonstrated that axolotl endothelial cells exhibit higher rates of macropinocytosis, a non-regulated type of transcellular transport.
CONCLUSIONS: Our study demonstrated that, under physiological conditions, the blood-brain barrier exhibited species-specific variations, including permeability threshold, blood vessel coverage, and macropinocytosis rate. Future studies are needed to test whether the higher permeability observed in salamanders could have metabolic and immunological consequences contributing to their remarkable regenerative capacity.
Fig. 1. Size-selective permeability analysis in the axolotl and Xenopus BBB. A-H Confocal images illustrating the Xenopus (A, C, E, G) and axolotl (B, D, F, H) vasculature labelled with isolectin. Red dashed lines delineate the brain midline. The arrow indicates the choroid plexus. All images are full z-projections of a confocal stack from cleared brains. Insets indicate the area of the brain. Scale bars = 100 µm. I Scheme of the experimental workflow to analyze the BBB permeability. J Scatter plot depicting the Manders’ coefficients obtained after injecting the 1, 3 and 70 kDa tracers in both axolotl and Xenopus. Data points were color-coded according to the species (blue: Xenopus; orange: axolotl). Each data point represents one animal and the statistical analysis was based on simple linear regression (Slope: pValue = 0.0878; Elevation: pValue < 0.0001). Abbreviations: ba: basilar artery; ltv: lateral telencephalic vein; moa: medial olfactory artery; pb: posterior branch of cerebral carotid; poa: preoptic artery; V: ventricle
Fig. 2. Analysis of the 3 and 70 kDa tracer permeability in the axolotl and Xenopus BBB. A-D’’ Confocal micrographs of cleared whole-mount Xenopus (A-A’’, hindbrain; C–C’’, midbrain) and axolotl (B-B’’, hindbrain; D-D’’, midbrain) CNS injected with the 3 kDa (A-B’’) or the 70 kDa (C-D’’) tracer together with isolectin. Low retention areas (3 kDa: B-B’’; 70 kDa: D-D’’) were indicated with orange arrowheads and high retention areas with orange arrows. All images are full z-projections of a confocal stack. Insets indicate the area of the brain. Scale bars = 100 µm. (E, F) Graphs depicting the Misolectin coefficients after 3 kDa (E) and 70 kDa (F) injections in Xenopus and axolotl. Each data point represents one animal. Data are shown as mean ± SEM and statistical analysis is based on Welch´s t-test (E; p-Value = 0.03) and Student´s t-test (F; p-Value = 0.12). Abbreviations: Ax: axolotl; V: ventricle; Xe: Xenopus
Fig. 3. Analysis of tracer uptake by the axolotl ECs. A Scheme of the experimental design analyzing the permeability of the larger tracers. B-C’’ Images illustrating the colocalization of the 3 kDa (B-B’’) and 70 kDa (C–C’’) tracers with isolectin+ blood vessels. D Scheme representing the experimental set-up utilized to assess the tracers EC uptake. E Confocal image of a transversal section of the axolotl hindbrain showing the location of the 70 kDa tracer after perfusion. F, F' are magnifications of the boxed area in (E). The orthogonal projections illustrated the localization of the 70 kDa tracers inside isolectin+ EC. All images are full z-projections of a confocal stack. G, H Internal axolotl EC surface (width ca. 8 × 12 µm, depth 16.4 µm). The surrounding EM plane shows a cell nucleus (right) and numerous profiles of neurites. Type of ruffles (1: blebs; 2: planar folds; 3: cup-shaped). Red arrows: macropinosomes. (I, J) EM reconstruction of a Xenopus EC luminal surface (width ca. 14 × 24 µm, depth 16 µm). Red arrowheads: extracellular vesicles. Scale bars in B, B’, B’’, C, C’, C’’ = 20 µm; E, F, F’’ = 100 µm; G, H, J = 2 µm; I = 5 µm
Fig. 4. Analysis of the 1 kDa tracer permeability in the Amphibian CNS. A Experimental design to analyze the BBB permeability. B-C’’ Micrographs of cleared whole-mounts Xenopus (B-B’’) and axolotl (C–C’’) telencephalon showing the distribution of the injected 1 kDa tracer. The vasculature was labeled using isolectin. D Graph indicating the Manders’ coefficients in the Xenopus (blue dots) and axolotl (orange dots) BBB. Each data point represents one animal. Data are shown as mean ± SEM and statistical analysis is based on Welch´s t-test (p-Value = 0.003). E Micrograph of a cleared whole-mount axolotl hindbrain injected with the 1 kDa tracer and isolectin after 10 min survival. Inset indicate the area of the brain. All images are full z-projections of a confocal stack. Scale bars = 100 µm. Abbreviations: Ax: axolotl; Xe: Xenopus
Fig. 5. Analysis of the permeability threshold in pre- and post-metamorphic Pleurodeles. A Scheme of the experimental workflow. B-C’’ Confocal images of cleared whole-mount telencephala in pre- (B-B’’) and post-metamorphic (C–C’’) Pleurodeles after co-injecting the 1 kDa tracer with isolectin. All images are full z-projections of a confocal stack. Insets indicate the area of the brain. Scale bars = 50 µm. D Dot-plot indicating the Manders’ coefficient for the 1 kDa tracer in Xenopus (blue dots), Pleurodeles pre-metamorphic (light green dots), Pleurodeles post-metamorphic (dark green dots) and axolotl (orange dots). Each data point represents one animal. Data are shown as mean ± SEM and statistical analysis is based on One-way ANOVA (pValue = 0.004) and Sidak’s multiple comparisons test (Xe vs. Pleurodeles Pre: pValue = 0.357; Xe vs. Pleurodeles Post: pValue = 0.555; Xe vs. Ax: pValue = 0.003; Pleurodeles Pre vs. Pleurodeles Post: pValue > 0.9999; Pleurodeles Pre vs. Ax: pValue = 0.102; Pleurodeles Post vs. Ax: pValue = 0.055). Abbreviations: Ax: axolotl; n.s: not significant; Pre: pre-metamorphic; Post: Post-metamorphic; Xe: Xenopus
Fig. 6. Claudin-5 expression pattern in Xenopus, Pleurodeles and axolotl. A-C’’ Confocal images of a transversal section of the Xenopus telencephalon (A, A’), Pleurodeles midbrain (B, B’) and axolotl telencephalon (C, C’) stained with DAPI, isolectin and Claudin-5. The orange arrowheads marked isolectin.+ immune cells and orange arrows shown Claudin-5 negative blood vessels. D, D’ Micrographs illustrating the axolotl testis labeled with DAPI, isolectin and Claudin-5. All images are full z-projections of a confocal stack. Scale bars = 50 µm. Abbreviations: Cldn5: Claudin-5
