Combining anatomical and cytobiological factors to assess the efficacy of ADSC-EVS in modulating retinal excitotoxicity

Tianqi Duan^1,2, Zhaolin Gao^1,3, Aixiang Luo¹, Jufang Huang¹

¹ Department of Anatomy and Neurobiology, School of Basic Medical Sciences, Central South University, Changsha, Hunan Province, China;
² Aier Eye Institute, Changsha, Hunan Province, China
³ Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan Province, China

Objective: Adipose mesenchymal stem cell-derived extracellular vesicles (ADSC-EVs) has emerged as a promising approach for treating glutamate-induced retinal excitotoxicity. However, the specific mechanisms underlying this therapeutic effect remain elusive. Previous studies have confirmed that ADSC-EVs play a protective role by modulating the phosphorylation of PKC and GluA2 in retinal ganglion cells (RGCs). This study aims to explore the anatomical and cytobiological factors contributing to the regulatory effects of ADSC-EVs.
Methods: We conducted experiments using Sprague Dawley (SD) rats and rat retinal neuronal precursor (R28) cells. The SD rats were anesthetized via intraperitoneal injection of 2% pentobarbital sodium. After modeling the rats via intravitreal injection of glutamate, we collected the eyeballs and subsequently euthanized the animals under anesthesia by cervical dislocation. Our experimental methods included the HE staining method, PI staining, TUNEL, electroretinography, small molecule RNA sequencing, RT-PCR assay, PI staining, Western-blot assay, and immunofluorescence assay.
Results: (1) ADSC-EVs rapidly entered the RGCs and effectively alleviated glutamate-induced inner retinal damage, including apoptosis in the ganglion cell layer and inner nuclear layer, a decrease in the thickness of inner plexiform layer, as well as reductions in the amplitudes of the a-, b-, and SOP-waves; (2) ADSC-EVs targeted and inhibited PLCD1 expression through miR-23a-5p. This in turn, regulated PKCA/GluA2 phosphorylation levels and GluA2 expression at the cell membrane.
Conclusion: ADSC-EVs exhibit the capacity to modulate excitotoxic damage to inner retinal anatomical structures in a short period of time through both direct and indirect means. In this regulatory process, miR-23a-5p plays an important role.

Keywords: Adipose mesenchymal stem cell-derived extracellular vesicles; excitotoxicity; inner retinal; miR-23a-5p.

Ethical statement: the Animal Ethics Committee of Central South University (No. CSU-2022 – 0088).

Funding statement: the Key Research and Development Program of Hunan Province, Nos. 2018SK2090 , 2022SK2079; the Human Resource Bank Program of Hunan Province, No. 2020TP3003; the School-Enterprise Joint Program of Central South University, No. 2021XQLH092.