To develop an endothelialized microfluidic chip model that simulates the spatial architecture and bioactivity of retinal vasculature, enabling thrombosis modeling and thrombolytic efficacy validation.

A tri-level microvascular network chip (300/200/100 μm diameters) with bifurcated architecture was fabricated using soft lithography. Human retinal microvascular endothelial cells (HRMECs) were perfused into channels, with endothelial coverage monitored via phase-contrast microscopy and F-actin staining. Cellular bioactivity was assessed using mitochondrial membrane potential probes (5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide, JC-1) and nitric oxide (NO) quantification. Fresh blood samples from 10 healthy donors (Yongchuan Hospital Affiliated to Chongqing Medical University, March to June 2024) were perfused with digital injection pump to mimic blood flow in human body into 3 experimental groups: normal whole blood, and TNF-α-activated endothelium+normal blood, TNF-α-activated endothelium+TNF-α-treated blood. Three inlet blood flow rates of 37.8、11.1 and 3.5 μL/min were set in each group. Two experimental groups, normal saline and recombinant human tissue-type plasminogen activator (rtPA), were established using the endothelialized microfluidic thrombosis model to validate thrombolytic efficacy. Endothelial functional impacts were assessed through integrated DAPI/NO staining and thrombosis model analysis across 3 intervention phases: pre-thrombosis, post-thrombosis, and post-thrombolysis.

A tri-level microfluidic vascular model (300/200/100 μm diameters) was successfully constructed. In 72 h after endothelial cell perfusion, complete channel coverage was achieved, with phase-contrast microscopy and F-actin staining confirming confluent cellular alignment. JC-1/NO assays validated preserved endothelial bioactivity. Compared with the whole blood group, both TNF-α-activated endothelium+normal blood and TNF-α-activated endothelium+TNF-α-treated blood groups exhibited significantly increased thrombus occupancy rates at identical flow rates (all P<0.001). Notably, TNF-α-activated endothelium+TNF-α-treated blood group demonstrated the highest thrombus ratio at 3.5 μL/min (P<0.001). The rtPA group showed superior thrombolytic efficacy versus saline (P<0.001). Endothelial monolayer integrity was maintained across intervention phases, with thrombosis triggering significant NO elevation (P<0.001).

Our retinal vasculature-mimetic microfluidic model enables precise thrombosis modeling and drug evaluation, providing new methodology for studying retinal vascular occlusive diseases.

To analyze the genetic features of congenital aniridia in a family and to explore the characteristics of PAX6 gene mutations and differences in clinical phenotypes.

The medical history and clinical data of this family line were collected. Whole-exon gene sequencing and data analysis were performed, and homology modelling server SWISS-MODEL was applied to construct the corresponding protein model for analysis.

All 4 individuals with onset in this family line had photophobia and difficulty in opening the eyes due to iris deficiency, with the same clinical phenotypes of aniridia, cataracts, and macular centro-concave dysplasia. But, there were still individual differences in phenotypes. Genetic examination showed that all 4 affected individuals were heterozygous for a heterozygous deletion shifted variant c.442_452del: p. M148Afs*48 on the PAX6 gene, which is highly conserved among species. Homozygous modeling suggested that the mutated PAX6 gene ultimately led to differential changes in protein conformation.

The newly identified PAX6 gene variant is not phenotypically identical among members of the family with congenital aniridia, and homology modeling analysis has increased the understanding that aberrant expression of PAX6 leads to structural abnormalities in the protein. Genetic examination may provide genetic evidence for this family line.