Low-Intensity Pulsed Ultrasound Attenuates Postoperative Neurocognitive Impairment and Salvages Hippocampal Synaptogenesis in Aged Mice

Abstract

Postoperative neurocognitive impairment is an urgent problem with global aging accelerating. The prevention and treatment of postoperative neurocognitive impairment have been widely investigated but lack effective strategies. Low-intensity pulsed ultrasound (LIPUS), a non-invasive tool, has shown an effect on neuroprotection, but whether it could attenuate the postoperative neurocognitive impairment and the underlying mechanisms remains unknown. An experimental setup for LIPUS stimulation of the hippocampus was well established. A laparotomy model in aged mice was applied, and a Morris water maze was used to assess cognitive function. RT-qPCR and western blotting were used to detect levels of Piezo1, synapse-associated proteins in the hippocampus, respectively. Immunofluorescent staining was also used to determine the neural activation and Piezo1 expression. The results showed that LIPUS increased synapse-related proteins of the hippocampus and attenuated cognitive impairment in aged mice. Meanwhile, LIPUS suppressed the overexpression of Piezo1 in the hippocampus. We further found that LIPUS promoted Calpain1 activity and increased extracellular regulated protein kinases (Erk) phosphorylation. Our results suggested that LIPUS could improve cognitive impairment and increase hippocampal synaptogenesis through the Piezo1-mediated Calpain1/ Erk pathway. LIPUS could be used as an effective physical intervention to alleviate postoperative cognitive dysfunction in the aged population.

Keywords: Calpain1; Piezo1; delayed neurocognitive recovery (dNCR); low-intensity pulsed ultrasound (LIPUS); synaptogenesis.

Figure 1

Overview of the LIPUS application. (A) Experimental design. (B) Diagram of the LIPUS setup and dorsal hippocampus positioning. (C) Acoustic intensity distribution of the lateral and axial direction was expressed by the spatial-peak pulse-average intensity (ISPPA). (D) Illustration of ultrasound stimulation parameters: fundamental frequency (FF) of 1 MHz, pulse repetition frequency (PRF) of 1 Hz, sonication duration (SD) of 50 ms, and duty cycle (DC) of 5%.

Figure 2

LIPUS can safely and precisely act on the dorsal hippocampus. Immunofluorescence staining of c-Fos (A) and HE stain (B) of the dorsal hippocampus with or without US treatment. CON: The sham group; US: The US treated group for 15 min.

Figure 3

LIPUS improves hippocampus-dependent learning and memory after anesthesia/surgery in aged mice. (A) Escape latency in the training stage before anesthesia/surgery. (B) Escape latency one day after LIPUS application. (C)Platform crossing times 1 day after anesthesia/surgery after LIPUS application. (D) Swimming speed of four groups during the process of testing. (E) The representative swimming trajectories of the four groups during the Morris water maze test. n = 12 per group. Data were presented as Mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 4

LIPUS attenuates neuroinflammation and synaptic dysfunction of the hippocampus after anesthesia/surgery in aged mice. The mRNA level of inflammatory cytokines of TNF-α (A), IL-1β (B), and IL-6 (C) in the hippocampus among the four groups (n = 6 per group). Western blotting analysis of the synapse-related proteins BDNF (D), Synaptophysin (E), and PSD95 (F) in the hippocampus among the four groups (n = 4 per group). Data were presented as Mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparisons test. * p < 0.05.

Figure 5

LIPUS inhibits Piezo1 expression after anesthesia/surgery in aged mice. (A) The mRNA expression of Piezo1 was qualified by RT-qPCR among the four groups (n = 6 per group). (B) Western blotting detection of Piezo1 among the four groups (n = 4 per group). (C) Immunofluorescence detection of Piezo1 expression in CA1 (C,D), CA3 (C,E), and DG (C,F) regions among the four groups (n = 3 per group). Data were presented as Mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparisons tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

LIPUS may modulate the calpain1/Erk pathway to salvage synaptogenesis after anesthesia/surgery in aged mice. Western blotting analysis of Calpain1 (A) and Calpian2 (B) expression among the four groups. (C) PHLPP, as the substrate of calpain1, was used to represent the activity of calpain1. (D) Erk phosphorylation level was expressed as the ratio of p-Erk to total Erk and normalized to the internal control β-actin. n = 4 per group. Data were presented as Mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparisons tests. * p < 0.05, **p < 0.01, *** p < 0.001.

Figure 7

The possible signaling pathway of LIPUS mediated neuroprotection after anesthesia/surgery in aged mice. Piezo1 expression was increased in aged mice undergoing anesthetic surgery, and an excessive influx of Ca²⁺ inhibited calpain1 activity, manifested as reduced PHLPP degradation, further alleviated Erk phosphorylation, and led to synapse dysfunction (left column). In contrast, LIPUS application inhibited Piezo1 expression and enhanced calpain1 activity, promoting PHLPP degradation, which released the inhibition of Erk phosphorylation and rescued synaptic function (right column).