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Updated: Jul 7, 2025
An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers
Published on: February 26, 2018
C-kit controls blood-brain barrier permeability by regulating caveolae-mediated transcytosis after chronic cerebral
Junkui Shang1, Wei Li1, Huiwen Zhang1
1Department of Neurology, Zhengzhou University People's Hospital, Henan Provincial People's Hospital, Zhengzhou, Henan 450003, China.
Insights
Chronic cerebral hypoperfusion causes blood-brain barrier dysfunction. C-kit receptor tyrosine kinase drives this by increasing transcytosis in endothelial cells, offering a therapeutic target for neurodegenerative diseases.
Area of Science:
- Neuroscience
- Cell Biology
- Pathology
Background:
- Blood-brain barrier (BBB) dysfunction is central to chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases.
- Endothelial cells (EC) form the BBB, but mechanisms of CCH-induced dysfunction remain unclear.
Purpose of the Study:
- To elucidate molecular mechanisms of EC-induced BBB dysfunction following CCH.
- To identify and validate C-kit as a key regulator of BBB integrity in CCH.
Main Methods:
- Immunostaining and transmission electron microscopy assessed BBB function.
- EC enrichment followed by RNA sequencing identified C-kit.
- Pharmacological inhibition (imatinib) and genetic downregulation (AAV-BR1-C-kit shRNA) of C-kit were used.
- C-kit activation with stem cell factor (SCF) was investigated.
Main Results:
- Macromolecular protein entry via EC transcytosis after CCH led to neuronal loss.
- Receptor tyrosine kinase C-kit was identified as a key EC dysfunction molecule.
- C-kit inhibition (imatinib, AAV-BR1-C-kit shRNA) reduced BBB leakage by decreasing caveolae-mediated transcytosis.
- SCF treatment increased BBB permeability by enhancing caveolae-mediated transcytosis.
Conclusions:
- C-kit is a critical regulator of BBB permeability in CCH.
- Targeting C-kit-mediated caveolae transcytosis offers a therapeutic strategy for CCH-related neurodegeneration.
Abstract:
Blood-brain barrier (BBB) dysfunction plays a pivotal role in the pathology of chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases. Continuous endothelial cells (EC) that line the blood vessels of the brain are important components of the BBB to strictly control the flow of substances and maintain the homeostatic environment of the brain. However, the molecular mechanisms from the perspective of EC-induced BBB dysfunction after CCH are largely unknown. In this study, the BBB function was assessed using immunostaining and transmission electron microscopy. The EC dysfunction profile was screened by using EC enrichment followed by RNA sequencing. After identified the key EC dysfunction factor, C-kit, we used the C-kit inhibition drug (imatinib) and C-kit down-regulation method (AAV-BR1-C-kit shRNA) to verify the role of C-kit on BBB integrity and EC transcytosis after CCH. Furthermore, we also activated C-kit with stem cell factor (SCF) to observe the effects of C-kit on BBB following CCH. We explored that macromolecular proteins entered the brain mainly through EC transcytosis after CCH and caused neuronal loss. Additionally, we identified receptor tyrosine kinase C-kit as a key EC dysfunction molecule. Furthermore, the pharmacological inhibition of C-kit with imatinib counteracted BBB leakage by reducing caveolae-mediated transcytosis. Moreover, treatment with AAV-BR1-C-kit shRNA, which targets brain EC to inhibit C-kit expression, also ameliorated BBB leakage by reducing caveolae-mediated transcytosis. Furthermore, the SCF increased the permeability of the BBB by actively increasing caveolae-mediated transcytosis. This study provides evidence that C-kit is a key BBB permeability regulator through caveolae-mediated transcytosis in EC after CCH.
