Elsevier

Neuroscience

Volume 86, Issue 4, 18 June 1998, Pages 1245-1257
Neuroscience

Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood–brain barrier breakdown in vivo

https://doi.org/10.1016/S0306-4522(98)00058-XGet rights and content

Abstract

The tight junctions found between cerebral vascular endothelial cells form the basis of the blood–brain barrier. Breakdown of the blood–brain barrier is a feature of a variety of CNS pathologies that are characterized by extensive leucocyte recruitment, such as multiple sclerosis and stroke. The molecular mechanisms associated with opening of the blood–brain barrier and leucocyte recruitment in vivo are currently poorly understood. We have used an in vivo rat model to investigate the molecular response of the CNS endothelium to neutrophil adhesion and migration. Injection of interleukin-1β into the striatum of juvenile brains results in a neutrophil-dependent increase in vessel permeability at 4 h. Only a subset of blood vessels were associated with neutrophil recruitment. These particular vessels displayed an increase in phosphotyrosine staining, loss of the tight junctional proteins, occludin and zonula occludens-1, and apparent redistribution of the adherens junction protein vinculin. Examination of these vessels under the electron microscope indicated that the cell–cell adhesions in such vessels are morphologically different from normal junctions.

This study provides the first direct evidence in vivo that leucocyte recruitment can trigger signal transduction cascades leading to junctional disorganization and blood–brain barrier breakdown. Our results have established an endothelial cell molecular profile associated with leucocyte-induced blood–brain barrier breakdown in vivo, and the relevance of different in vitro cell culture models may now be viewed more objectively.

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Experimental animals

Male Lewis rats aged either three weeks (30–40 g, juvenile) or 12 weeks (>300 g, adult) were obtained from Harlan-OLAC (Bicester, Oxfordshire, U.K.). All animals were housed under standard conditions, with pelleted food and water available ad libitum.

Stereotaxic surgery

Stereotaxic surgery was performed as described previously.[36]Briefly, anaesthetized rats were held in a stereotaxic frame and the skull and brain exposed. One hundred units of human recombinant interleukin-1β (IL-1β; juveniles: n=12; adults: n=6) or

Interleukin-1β induces neutrophil-dependent blood–brain barrier breakdown in juvenile, but not adult, rats

One hundred units of human recombinant IL-1β induced neutrophil adhesion to blood vessels and BBB breakdown when injected into the striatum of juvenile, but not adult, rats. The tracer HRP is normally excluded from the brain by the BBB, but injected juvenile animals showed extensive HRP accumulation throughout the tissue of the injected hemisphere (Fig. 1D). This breakdown of the BBB in the juvenile animal is wholly neutrophil dependent,[5]allowing us to dissect out the response of the

Discussion

We have examined, for the first time in vivo, the molecular events which occur within the cerebral vascular endothelium during neutrophil-dependent BBB breakdown. We describe data which show that the cerebral vascular endothelium is an active participant in the recruitment of neutrophils to the brain parenchyma. A generalized reponse of the endothelial cells to injection of IL-1β was the up-regulation of ICAM-1, seen in most of the vasculature within the injected hemisphere. A more specific

Conclusion

Our knowledge of the events that occur in vivo during BBB breakdown is still very limited. The data presented in this paper have demonstrated, for the first time in vivo, alterations in both signalling and junctional components during cell-mediated BBB breakdown. Interestingly, only a particular subset of vessels within the brain will permit leucocyte migration, although it is not known why only these vessels become the targets for leucocyte adhesion and migration. We have begun to generate a

Acknowledgements

We would like to thank Dr M. Itoh for the anti-ZO-1 antibody and Dr K. Miller for the anti-VE-cadherin antibody. We also thank Sara Fearn and Mandy Townsend for technical assistance. This work was supported by the Medical Research Council.

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