Neuroinflammation, blood-brain barrier dysfunction, and cognitive decline in pulmonary arterial hypertension: an experimental study

Figures

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  Fig. 1. Echocardiographic parameters. (A) Pulmonary Artery Systolic Pressure. (B) PAT/PET ratio. PAT: pulmonary acceleration time; PET: pulmonary ejection time. (C) Echocardiographic representative images, yellow outline, peak blood flow in the pulmonary artery. (D) Left ventricular ejection fraction. (E) Right ventricular hypertrophy index calculated as the ratio of the right ventricular weight to the combined weight of the left ventricle and septum (RV/LV + S), adjusted for body weight. CTRL: control; PAH: pulmonary arterial hypertension. Data are presented as means ± SD. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 2. (A) Percentage of perivascular collagen deposition. (B) Representative images of perivascular collagen in pulmonary arteries (in blue). (C) Quantification of vessel wall thickness. (D) Representative immunohistochemistry images of α-smooth muscle actin (in brown). Data are presented as means ± SD. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 3. (A) Rotarod Test. Latency to fall (seconds). (B) Open field test. Total distance traveled (meters) (C) Center entries (D) Center time in seconds (E) Center distance (meters) (F) Periphery distance (meters) (G) Ratio center/periphery. (H) Representative track plot reports recorded during the 10 min test sessions. (I) Correlation between PASP and total distance traveled was assessed using Spearman’s correlation. A negative correlation was observed (r = − 0.467, p = 0.021). CTRL: control. PAH: pulmonary arterial hypertension. Data are presented as means ± SD. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 4. Novel object recognition test. Time in percentage exploring familiar or new objects. CTRL: control. PAH: pulmonary arterial hypertension. The results were expressed as the percentage of time exploring each object and analyzed according to the mean exploration time for each object with the fixed value of 50% (chance level). Data are presented as means ± SD. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 5. Immunofluorescence of microglia (Iba-1+) in cortex (A) and (B) and hippocampus (C) and (D) at D15 at 20× magnification. (A) Number of Iba-1 + cells in cortex; (B) Mean microglial area in cortex; (C) Number of Iba-1 + cells in hippocampus; (D) Mean microglial area in hippocampus. (E) Representative images obtained of DAPI and Iba-1+, only Iba-1+, and only Iba-1 + enlarged. Scale bar: 100 μm. Enlarged images obtained at 100x magnification. Scale bar: 20 μm. CTRL: control; PAH: pulmonary arterial hypertension. Data are presented as mean ± SD, with individual data points representing independent animals. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 6. Immunofluorescence of astrocytes (GFAP+) in cortex (A) and (B) and hippocampus (C) and (D) at D15 at 20× magnification. (A) Number of GFAP+ cells in cortex; (B) Mean microglial area in cortex; (C) Number of GFAP+ cells in hippocampus; (D) Mean microglial area in hippocampus. (E) Representative images obtained of DAPI and GFAP+, only GFAP+, and only GFAP+ enlarged. Scale bar: 100 μm. Enlarged images obtained at 100x magnification. Scale bar: 20 μm. CTRL: control; PAH: pulmonary arterial hypertension. Data are presented as mean ± SD, with individual data points representing independent animals. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 7. (A)–(D) Sholl analysis showing the number of intersections as a function of radial distance from the soma in cortical astrocytes. (A) Cortical astrocytes (B) CA1 astrocytes (C) microglial cortex (D) microglial hippocampus from control (CTRL) and pulmonary arterial hypertension (PAH) groups. (E)–(H) Quantification of maximum Sholl radius, reflecting total process extension, revealed a significant increase in cortical (E) and CA1 (F) astrocytes from the PAH group, while no differences were observed in cortical (G) or CA1 microglia (H). Data are presented as mean ± SD, with individual data points representing single cells. Comparisons were performed using two-way ANOVA followed by Tukey multiple comparisons for Sholl profiles and Mann–Whitney tests for maximum radius comparisons (p < 0.05).

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  Fig. 8. (A)–(B) Representative immunofluorescence images of NeuN-positive neurons (green) and DAPI nuclear staining (blue) in the cortex (A) and hippocampus (B) of control (CTRL) and pulmonary arterial hypertension (PAH) groups. (C)–(D) Quantification of NeuN-positive cells in the cortex (C) and hippocampus (D). No significant diference in neuronal density was observed in the cortex between CTRL and PAH groups. In contrast, a significant reduction in NeuN-positive cells was detected in the hippocampus of PAH animals compared to CTRL (p = 0.019). Data are presented as mean ± SD, with individual data points representing independent animals. Comparisons made using the Unpaired t test (p < 0.05).

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  Fig. 9. (A) Representative immunofluorescence images showing extravascular IgG immunofluorescense (green) in the cortex of control (CTRL) and pulmonary arterial hypertension (PAH) animals, indicating blood brain barrier (BBB) leakage. Scale bar: 100 μm. (B) Quantitative analysis of IgG fluorescence intensity outside blood vessels revealed a significant increase in the PAH group compared to controls. Data are presented as median and interquartile range (Q1–Q3), with individual data points representing independent animals. Comparisons were performed by Mann–Whitney test (p < 0.05).

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  Fig. 10. Representative immunofluorescence images of CTRL and PAH groups showing individual fluorescence channels at 60× magnification. Scale bar: 20 μm. Nuclei are labeled with DAPI (blue), astrocytes with GFAP (green), and vascular structures with CD31/PECAM-1 (red). Images illustrate the distribution of each marker in the cortex. Merged images illustrate the spatial relationship between astrocytic processes and blood vessels in CTRL and PAH animals.

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  Fig. 11. Quantification of BNP, TNF-α, and IL-1β in lung, plasma, cortex, and hippocampus measured by ELISA. Data are presented as median with interquartile range (Q1-Q3). Comparisons were performed using the Unpaired t test or Mann-Whitney test (p < 0.05).