Safety of ultrasound combined with microbubbles irradiation on rat retina and its threshold

Objective  To explore the safety of ultrasound combined with microbubbles (USMB) irradiation on the rat retina, and to preliminarily identify the USMB parameter threshold that causes irreversible damage to the retina.

Methods  Eighty-four Sprague-Dawley rats were randomly divided into three groups according to the mechanical index (MI) of contrast-enhanced ultrasound (CEUS): MI=0.2, 0.4, and 0.8. CEUS, fundus fluorescein angiography (FFA), optical coherence tomography (OCT), electroretinogram (ERG), and hematoxylin-eosin (HE) staining were used to detect changes in retinal structure and function before USMB irradiation and at different time points after irradiation. Real time fluorescence quantitative PCR was used to detect the relative mRNA expression levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the rat retina. Another six normal Sprague-Dawley rats were selected as baseline controls and underwent ERG, HE staining, and real-time fluorescence quantitative PCR. All animal procedures in this study strictly followed the Association for Research in Vision and Ophthalmology Statement. All animal experimental protocols have been approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (No. AMUWEC20245237).

Results  When MI=0.2, the peak intensity (PI) and area under the curve (AUC) values in the time-intensity curve immediately after USMB irradiation were 83.500±1.310 and 4 560.41±143.10, respectively, which were significantly higher than 71.600±0.778 and 4 209.20±93.08 before USMB irradiation (all P<0.01). No statistically significant differences in other research indicators were observed at every time point after USMB irradiation compared to before USMB irradiation (all P>0.05). When MI=0.4, the PI and AUC values immediately after USMB irradiation significantly decreased compared to before USMB irradiation (both P<0.05). FFA results showed a slight leakage immediately after USMB and disappeared 1 day after USMB irradiation. OCT and HE staining showed mild edema of the retina on day 1 after USMB irradiation, with an increase in inner and full retinal thickness compared to before USMB irradiation, and the differences were statistically significant (all P<0.05). The ERG results showed that the amplitudes of a wave, b wave, and oscillatory potential (OPs) of dark adaptation 3.0 at 1 day after USMB irradiation was significantly reduced compared to before USMB irradiation (all P<0.05). The relative expression levels of IL-1β, IL-6, and TNF-α mRNA in the retina 1 day after USMB irradiation were significantly increased compared to before USMB irradiation (all P<0.05). When MI=0.8, the PI and AUC values immediately, 1 day, 3 days, and 7 days after USMB irradiation were significantly reduced compared to before USMB (all P<0.01). FFA showed immediate large vessel leakage after USMB irradiation, which persisted until 1 day after USMB irradiation. OCT and HE staining showed severe retinal edema at 1 and 3 days after USMB irradiation, with significant increases in inner and full layer retinal thickness compared to before USMB irradiation, and the differences were statistically significant (all P<0.01). At 7 days after USMB irradiation, the retina atrophied, and the inner and full layer retinal thickness decreased significantly compared to before USMB irradiation, and the differences were statistically significant (all P<0.05). The ERG results showed that at 1, 3, and 7 days after USMB irradiation, the amplitudes of a wave, b wave, and OPs of dark adaptation 3.0 were significantly reduced compared to before USMB irradiation (all P<0.05). The relative expression levels of IL-1β and TNF-α mRNA in the retina at 1 and 3 days after USMB irradiation were significantly higher than before USMB irradiation, and the relative expression level of IL-6 mRNA in the retina at 1 day after USMB irradiation was significantly higher than before USMB irradiation (all P<0.01).

Conclusions  When MI is below 0.4, USMB irradiation has no effect on the structure and function of rat retina or the effect is reversible. When MI=0.8, USMB irradiation causes irreversible ischemic damage to the structure and function of rat retinas.