The Blood-Labyrinth Barrier (BLB) is characterized by a complex of interstitial fluid junctions critical for regulating the exchange of substances between the cochlear blood and intercellular fluid (Shi X et al., 2022; Noman A et al., 2022). Its fundamental role involves protecting the inner ear from potentially noxious agents in the bloodstream, while concurrently facilitating the selective translocation of ions, fluids, and nutrients vital for cochlear functionality. This function underscores the BLB's integral contribution to the maintenance of cochlear homeostasis (Juhn SK et al., 2001; Gu J et al., 2022). Additionally, numerous studies have highlighted that the BLB is essential for normal hearing, functioning as a physiological, transport, and metabolic barrier (Sekulic M et al., 2023; Neng L et al., 2015). Pathological alterations in the BLB's permeability are increasingly recognized, not merely as a peripheral phenomenon but as a central mechanism underlying the pathogenesis of noise-induced and drug-induced hearing impairments. These findings suggest that the disruption in BLB integrity extends beyond the conventional paradigms of hair cell and spiral ganglion cell loss (Cosentino A et al., 2023; Wu J et al., 2017). Consequently, the BLB has emerged as a focal point for developing preventive and therapeutic strategies against sensorineural and neural auditory deficits, signaling a paradigm shift in the understanding and management of hearing loss conditions.

In 1998, Suzuki M et al. revealed that endothelial cell transport within the cochlea shows increased activity during the developmental stages of rats, specifically at 4, 7, and 11 days postnatal, with the maturation of the BLB being conclusively observed at 14 days post-birth (Suzuki M et al., 1998). Subsequent studies showed that in C57BL/6 mice, cochlear blood vessels do not achieve fully developed structural integrity by the 15th day of gestation, reaching full maturity by the 15th postnatal day (Iwagaki T et al., 2000). More recent research has detailed structural transformation within the stria vascularis (SV) of the BLB across different ages in C57BL/6 mice, from 1 to 21 months. These studies identified a reduction in capillary density beginning at 6 months, with pronounced capillary degeneration observable between 9 to 21 months. The reduction in capillary density was found to be closely linked to decreased levels of perivascular cells (PCs) and perivascular macrophage-like melanocytes (PVM/Ms) (Neng L et al., 2015). Due to limitations in research methodologies and animal models, the lack of comprehensive reports on the BLB's developmental mechanisms highlights the critical need for more detailed future investigations in this area.

The functional integrity of the BLB is maintained by a sophisticated network of endothelial cells, with its barrier properties significantly influenced by the orchestration of tight junctions and transcellular vesicular transport mechanisms, particularly transcytosis (Saunders NR et al., 2012; Siegenthaler JA et al., 2013). Tight junctions are paramount in moderating the permeability of substances across the barrier, forming a continuous seal among endothelial cells to restrict unauthorized passage (Saeki T et al., 2020). A reduction in the expression of tight junction proteins is directly correlates with increased permeability of the SV, thereby undermining the BLB's structural and functional integrity (Zhang J et al., 2020; Deng S et al., 2021). Simultaneously, the transcytosis mechanism plays a critical role in the regulated transfer of materials across the barrier, ensuring that transport rates remain tightly controlled to preserve barrier selectivity. Although vesicular transport within the BLB has received relatively scant attention in existing literature, available studies suggest that such vesicular dynamics are essential in facilitating the functional role of pericyte cells within the barrier matrix (Ghelfi E et al., 2018). This highlights the multifaceted nature of BLB regulation, underscoring the need for an integrated understanding of both tight junction and vesicular components to maintain barrier efficacy.

The gene Mfsd2a, known for its crucial role in transporting unsaturated fatty acids, has become a key focus in studies on endothelial barrier integrity. Research from 2014 revealed that Mfsd2a is essential in preserving the Blood-Brain Barrier (BBB) integrity by potentially inhibiting endocytosis in the endothelial cells of the central nervous system (Ben-Zvi A et al., 2014). Later study indicated that overexpression of Mfsd2a within the Blood-Retinal Barrier (BRB) could counteract the endothelial and retinal tissues endocytosis triggered by Wnt signaling pathways (Wang Z et al., 2020). Additionally, the role of Mfsd2a in facilitating trophoblastic fusion and the developmental intricacies of the placental barrier have been documented (Toufaily C et al., 2013; Yu HT et al., 2024). In neurological contexts, the absence of Mfsd2a correlates with increased vascular permeability and compromised BBB integrity, as evidenced by elevated transcytosis rates in the endothelial cells of both mice and zebrafish models (O'Brown NM et al., 2019; Li X et al., 2023). Conversely, Mfsd2a's lipid transport functionality is instrumental in maintaining a lipid-rich environment, which in turn reduces vacuole formation and limits endothelial transcytosis (Andreone BJ et al., 2017). The consensus across various studies highlights Mfsd2a's pivotal role in modulating barrier permeability and mediating substance transport processes. Although the BLB and the BBB share functional similarities, including similar capillary cross-sections and cellular compositions (Nyberg S et al., 2019), the exploration of Mfsd2a's contributions to the BLB's formation and operational dynamics remains limited. This is attributed to the BLB's increased complexity and reduced permeability relative to the BBB.

In this study, we hypothesize that Mfsd2a plays a crucial role in both the development and sustained functionality of the BLB. To investigate the maturation of the BLB in murine models, we utilized various fluorescent tracers, which confirmed that BLB integrity becomes evident by the developmental milestone of two weeks postnatal. Complementary in vitro assays involving stria vascularis endothelial cells supported our hypothesis, showing that a reduction in Mfsd2a expression correlates with increased BLB permeability. Additionally, our in vivo applications of Mfsd2a inhibitors led to a noticeable permeation of fluorescent tracers across the established BLB. Preliminary investigations also sought to determine Mfsd2a's influence on BLB functionality, specifically its impact on the expression of tight junction proteins in mice and its role in modulating endothelial cell transcytosis. Our findings indicate that Mfsd2a underpins the integrity of tight junction proteins; conversely, a decrease in Mfsd2a expression compromises these junctions and facilitates increased transcytosis across the BLB. To our knowledge, this study represents the first endeavor to elucidate Mfsd2a's importance in both establishing and maintaining the BLB, highlighting its critical role in ensuring the BLB's functional integrity. These insights position Mfsd2a as a promising target for therapeutic intervention aimed at regulating the BLB. Moreover, this research paves the way for utilizing Mfsd2a as a vehicle for targeted drug delivery, offering a novel approach to the treatment of hearing loss.