# In Vitro Comparison of Two Python‐Based Programs for the Automated Analysis of Tight‐Junction Phenotype in Brain Endothelium During Bacterial Infection

## TL;DR

This study compares two Python-based programs for analyzing tight junctions in brain endothelial cells during bacterial infection, showing their effectiveness and unique benefits.

## Contribution

The study provides a direct comparison of JAnaP and IJOQ for tight junction analysis, highlighting their distinct advantages in different contexts.

## Key findings

- JAnaP and IJOQ both effectively quantify tight junction disruption in brain endothelial cells.
- JAnaP provides detailed cellular-level data, while IJOQ reduces user time and bias.
- Both programs are suitable for studying blood–brain barrier dysfunction and CNS diseases.

## Abstract

Tight junction complexes are crucial features of brain endothelial cells, as they restrict the paracellular route across the blood–brain barrier. Tight junction disruption has been observed in conjunction with numerous diseases of the CNS. In such cases, the organization or integrity of cell–cell junctions may be analyzed with a variety of automated computer programs that quantitatively assess junction images. Here, we directly compare two previously developed python‐based programs—JAnaP and IJOQ— for the semi‐ or fully automated analysis of tight junctions in human stem cell‐derived brain‐like endothelial cells. Cells were infected with S. pneumoniae and S. agalactiae to initiate junction disruption, and occludin and ZO‐1 were analyzed in mock and infected groups via JAnaP and IJOQ. JAnaP and IJOQ both yielded comparable results for the quantification of tight junction disruption in brain endothelial cells. While JAnaP rendered data at the cellular level and gave more information regarding junction phenotype, IJOQ significantly reduced user time and eliminated potential user bias. Our results suggest that JAnaP and IJOQ are both appropriate for quantifying tight junction integrity in brain endothelial cells, and both may offer distinct advantages depending on their context of use.

Tight junction disruption is a hallmark of blood–brain barrier dysfunction. Thus, the quantification of tight junction integrity is important for understanding the mechanisms of BBB and CNS pathologies.Our comparison of two automated cell–cell junction analysis programs highlights the utility of both programs as a means of studying tight junction disruption in brain endothelial cells.Additionally, this study demonstrates the advantages of each program and suggests contexts in which one or the other may be more appropriate or beneficial.By evaluating the tools available for tight junction analysis, this study will promote further investigation of blood–brain barrier disruption and CNS disease.

Tight junction disruption is a hallmark of blood–brain barrier dysfunction. Thus, the quantification of tight junction integrity is important for understanding the mechanisms of BBB and CNS pathologies.

Our comparison of two automated cell–cell junction analysis programs highlights the utility of both programs as a means of studying tight junction disruption in brain endothelial cells.

Additionally, this study demonstrates the advantages of each program and suggests contexts in which one or the other may be more appropriate or beneficial.

By evaluating the tools available for tight junction analysis, this study will promote further investigation of blood–brain barrier disruption and CNS disease.