Amide proton transfer (APT) imaging-based study on the correlation between brain pH and voltage-gated proton channels in piglets after hypoxic-ischemic brain injury

Abstract

Background: The normal regulation of brain pH is particularly critical for protein structure and enzymatic catalysis in the brain. This study aimed to investigate the regulation mechanism of brain pH after hypoxic-ischemic brain injury (HIBI) through the combination of amide proton transfer (APT) imaging, the analysis of brain pH levels, and the analysis of voltage-gated proton channel (Hv1) expression in piglets with HIBI.

Methods: A total of 59 healthy piglets (age range, 3-5 days after birth; body weight, 1-1.5 kg) were selected. Six piglets were excluded due to death, modeling failure, or motion artifacts, leaving a total of 10 animals in the control group and 43 animals in the HIBI model group. At different time points (0-2, 2-6, 6-12, 12-24, 24-48, and 48-72 hours) after HIBI, brain pH, Hv1 expression, and APT values were measured and analyzed. The statistical analysis of data was performed using the independent samples t-test, analysis of variance, and Spearman rank correlation analysis. A P value less than 0.05 indicated statistical significance.

Results: As shown by the immunofluorescent staining results after HIBI, Hv1 protein expression in the basal ganglia reached a peak value at 0-2 hours, with a statistically significant difference between 0-2 hours and other time points (P<0.001). In piglets, the APT value reached a trough at 0-2 hours after HIBI, and subsequently, it gradually increased, and there was a significant difference between the control group and all HIBI model subgroups (P<0.001) except for the 2-6 hours subgroup (P=0.602). Brain pH decreased after HIBI and reached a trough at 0-2 hours, then gradually increased. Hv1 protein expression, pH, and APT values were all correlated (P<0.001).

Conclusions: After HIBI, values of brain pH, APT, and the expression of Hv1 changed over time and had a linear correlation. This suggests that there was a shift in brain hydrogen ions (H⁺) in the neural network and a change in brain pH after hypoxic-ischemic (HI) injury.

Keywords: Amide proton transfer (APT); brain; hypoxic-ischemic injury; pH; voltage-gated proton channels (Hv1).

Figure 1

Schematic diagram of pH measurement. The region of interest (basal ganglia) is marked with a yellow box. The experimental animal was kept lying prostrate on the operation bench, a pH electrode was vertically inserted 2.0 to 2.5 cm into the brain from the vertex to measure pH levels twice in the bilateral basal ganglia, and then the two measurement results were averaged.

Figure 2

Time-based change trend of APT values after HIBI. After hypoxic-ischemic brain injury, the APT value was decreased and then increased, and it reached a trough at 0–2 hours. There was a significant difference between the 0–2 hours subgroup and all the remaining subgroups, except for the 2–6 hours subgroup (P=0.602). APT, amide proton transfer; APTw, amide proton transfer weighted; HIBI, hypoxic-ischemic brain injury.

Figure 3

Immunofluorescent Hv1 expression after hypoxic-ischemic brain injury (400×). Lines 1 to 4 show Hv1 expression in the control group and at 2–6, 6–12, and 24–48 hours after hypoxic-ischemic brain injury. The left image shows Hv1 expression in the cell membrane, the middle image shows target proteins in the nucleus and cell membrane, and the right image shows the nucleus. The Hv1 expression peak was found at 2–6 hours after hypoxic-ischemic brain injury.

Figure 4

Violin plot for the time-based changes of Hv1 expression and pH after hypoxic-ischemic brain injury. After hypoxic-ischemic brain injury, Hv1 expression tended to increase first, then decrease, and reached a peak at 2–6 hours, whereas pH had an opposite change trend and reached a trough at 0–2 hours. Subsequently, it increased gradually. HIBI, hypoxic-ischemic brain injury.

Figure 5

Time-based change trends and correlations among the APT value, Hv1 expression, and pH after hypoxic-ischemic brain injury. After hypoxic-ischemic brain injury, there was a significant negative correlation between the APT value and Hv1 expression (ρ=–0.727; P<0.001) and between pH and Hv1 expression (ρ=–0.666; P<0.001), but a linear positive correlation between the APT value and pH (ρ=0.862; P<0.001). APT, amide proton transfer; APTw, amide proton transfer weighted.