Elsevier

Brain Research

Volume 969, Issues 1–2, 18 April 2003, Pages 59-69
Brain Research

Research report
Radiation-induced permeability and leukocyte adhesion in the rat blood–brain barrier: modulation with anti-ICAM-1 antibodies

https://doi.org/10.1016/S0006-8993(03)02278-9Get rights and content

Abstract

We assessed the acute effects of radiation on the rat blood–brain barrier. A cranial window model and intravital microscopy were used to measure changes in permeability and leukocyte adhesion in pial vessels after a localized, single dose of 20 Gy. Permeability was assessed using five sizes of fluorescein isothiocyanate (FITC)-dextran molecules (4.4-, 10-, 38.2-, 70-, and 150-kDa) with measurements performed before and 2, 24, 48, 72 and 96 h after irradiation for the 4.4 and 38.2-kDa molecules and before and 24 h after irradiation for the other three molecules. To demonstrate the nature of blood–brain barrier permeability, we concurrently studied the permeability of microvessels in the cremaster muscle. In both tissues, permeability to FITC-dextran was significantly greater 24 h after irradiation than before (P<0.05). The exception was that radiation did not affect the permeability of pial vessels to the 150-kDa molecule. The particle-size dependence of the permeability changes in the brain were indicative of altered integrity of endothelial tight junctions and occurred concomitantly with an increase in cell adhesion which was determined by fluorescent labeling of leukocytes with rhodamine 6G. An early inflammatory response to irradiation was apparent in the brain 2 h after irradiation. The numbers of rolling and adherent leukocytes increased significantly and peaked at 24 h. Injection with the anti-ICAM-1 mAb significantly reduced leukocyte adhesion and permeability thereby linking the two processes. These findings provide a target to reduce radiation-related permeability and cell adhesion and potentially the side effects of radiation in the CNS.

Introduction

Radiation therapy (RT) is one of the most effective treatments for tumors of the central nervous system (CNS). Despite recent advances in focal radiation delivery, the therapeutic benefit of RT is still limited by the ability of normal brain tissue to tolerate this treatment. Radiation alters the structure and function of microvascular networks [28], [38]. The microenvironment of the brain is regulated and protected by specific barriers, which include the vascular–endothelial barrier (blood–brain barrier [BBB]) at the capillary–parenchyma interface and the epithelial barrier (blood–cerebrospinal fluid barrier) at the choroid plexus [9]. The BBB is more than a physical barrier; it plays a fundamental role in regulating the movement of substances between the blood and the CNS. When the barrier between the vascular supply of the brain and the CNS parenchyma is disrupted, excess extravasation of proteins, biologic-response molecules (e.g. growth factors, cytokines, and clotting factors), inflammatory cells, and therapeutic drugs can damage the brain [4], [9], [24], [40]. The long-term sequelae of cranial radiation and its effect on hearing loss, cognitive dysfunction, and memory deficits are well documented [20], [41].

The endothelial cells that make up the BBB contain few pinocytotic vesicles and adhere to each other via tight junctions, which are formed by junction-specific proteins such as occludin. The amount of occludin in brain endothelial cells is at least five times as great as that in endothelial cells of other tissues [14]. Tight junctions limit paracellular transport of hydrophilic compounds into the CNS. This would manifest itself in a molecular size-dependent permeability or restricted diffusion [26]. Astrocytes in close proximity to the endothelial cells add another impediment to paracellular transport by biochemically conditioning the endothelial cells and strengthening the tight junctions between them [39]. In contrast to the endothelial cells of the BBB, those of the peripheral blood vessels possess very few tight junctions and contain more pinocytotic vesicles; peripheral vessels also lack the structural and biochemical support of astrocytes. Therefore, peripheral vessels are less resistant to the extravasation of substances from the vessel into surrounding tissue.

Radiation affects leukocyte–endothelial cell interactions within the blood vessels [29]. Rolling and adhesion are the first steps of extravasation of leukocytes into perivascular tissue. The cross-linking of adhesion molecules triggers cytoskeletal remodeling in endothelial cells, and this remodeling makes possible the transendothelial migration of the leukocytes [10], [11]. Intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed in the brain by endothelial cells and is upregulated by inflammatory stimuli [42]. Radiation increases the expression of adhesion molecules and cytokines. In vitro and in vivo studies have shown that ICAM-1 plays an important role in the increased adhesion of leukocytes to irradiated endothelium [1], [13], [17], [31], [34]. Vascular permeability and cellular interactions are probably interrelated and may be responsible for many of the early and late effects of RT observed in normal tissues. We speculated that blocking ICAM-1 expression on the endothelial cell surface would interfere with the rate and kinetics of the blood–brain barrier permeability caused by cranial radiation.

We used the closed cranial window model to characterize acute changes in the permeability of the cerebral pial microvessels after a single dose of ionizing radiation and compared these changes with those in the peripheral microvessels of the cremaster muscle. This comparison of the brain and peripheral vasculature and their respective acute responses to radiation will increase our knowledge of how radiation induces damage of healthy brain tissue. We measured the leukocyte–endothelial cell interaction as a function of time after irradiation, and tested the hypothesis that radiation-induced increase in permeability and cell adhesion can be modulated (suppressed) by blocking cell adhesion using anti-ICAM-1 mAb.

Section snippets

Animals

The 57 male Sprague–Dawley rats (8–9 weeks of age) used in this study were obtained from Harlan Laboratories (Frederick, MD, USA). All of the protocols included in this study followed the animal care guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center, Memphis. Sterile techniques were used during all surgical procedures.

Effect of radiation on the permeability of the BBB

A single dose of 20 Gy significantly increased the permeability of the BBB to 4.4-, 10-, 38.2-, and 70-kDa FITC-dextran, an effect that peaked 24 h after treatment. Before irradiation, the permeability of the BBB to 4.4-kDa FITC-dextran was 1.42±0.22×10−6 cm/s; 24 h later, it was 2.17±0.16×10−6 cm/s (P=0.013) (Fig. 2A). The permeability of the BBB to 38.2-kDa FITC-dextran was significantly higher 24 h after irradiation (2.33±0.33×10−7 cm/s) than before irradiation (1.06±0.14×10−7 cm/s; P=0.033)

Discussion

Radiation increased the permeability of pial venules in a distinct, particle-size-dependent manner, a finding that reflects the alteration of the tight junctions in the endothelial cells that contribute to the blood–brain barrier. Radiation-induced permeability was accompanied by an increase in leukocyte adhesion. Both processes were modulated by administration of anti-ICAM-1 monoclonal antibodies. Two mechanisms account for the extravasation of FITC-dextran (a hydrophilic substance) across the

Acknowledgements

The authors would like to thank Angela J. McArthur, Ph.D., for scientific editing. This research was supported in part by a Cancer Center Support Grant P30 CA 21765 from the National Institutes of Health and by the American Lebanese Syrian Associated Charities (ALSAC).

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