The use of hyperosmolar agents (osmotherapy) has been a major treatment for intracranial hypertension, which occurs frequently in brain diseases or trauma. However, side-effects of osmotherapy on the brain, especially on the blood–brain barrier (BBB) are still not fully understood. Hyperosmolar conditions, termed hyperosmolality here, are known to transiently disrupt the tight junctions (TJs) at the endothelium of the BBB resulting in loss of BBB function. Present techniques for evaluation of BBB transport typically reveal aggregated responses from the entirety of BBB transport components, with little or no opportunity to evaluate heterogeneity present in the system. In this study, we utilized potentiometric-scanning ion conductance microscopy (P-SICM) to acquire nanometer-scale conductance maps of Madin–Darby Canine Kidney strain II (MDCKII) cells under hyperosmolality, from which two types of TJs, bicellular tight junctions (bTJs) and tricellular tight junctions (tTJs), can be visualized and differentiated. We discovered that hyperosmolality leads to increased conductance at tTJs without significant alteration in conductance at bTJs. To quantify this effect, an automated computer vision algorithm was designed to extract and calculate conductance components at both tTJs and bTJs. Additionally, lowering Ca 2+ concentration in the bath facilitates tTJ disruption under hyperosmolality. Strengthening tTJ structure by overexpressing immunoglobulin-like domain-containing receptor 1 (ILDR1) protein abrogates the effect of hyperosmolality. We posit that osmotic stress physically disrupts tTJ structure, as evidenced by super-resolution microscopy. Findings from this study not only provide a high-resolution view of TJ structure and function, but also can inform current osmotherapy and drug delivery strategies for brain diseases.
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Multiscale imaging of corneal endothelium damage and Rho-kinase inhibitor application in mouse models of acute ocular hypertension
We developed a multiscale optical imaging workflow, integrating and correlating visible-light optical coherence tomography, confocal laser scanning microscopy, and single-molecule localization microscopy to investigate mouse cornea damage from the
- NSF-PAR ID:
- 10487571
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 15
- Issue:
- 2
- ISSN:
- 2156-7085
- Format(s):
- Medium: X Size: Article No. 1102
- Size(s):
- ["Article No. 1102"]
- Sponsoring Org:
- National Science Foundation
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Abstract Background Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet‐to‐be‐fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho‐associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown.
Methods Three‐dimensionally engineered dystrophin‐deficient
mdx skeletal muscles andmdx mice were used to test the role of ROCK in DMD muscle functionin vitro andin situ , respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generatingArhgef3 knockoutmdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild‐type or GEF‐inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine.Results Inhibition of ROCK with Y‐27632 improved muscle force production in 3D‐engineered
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