The breakdown of the BBB and the presence of CCL2 both have a role in the initiation of disease signs in EAE and MS. Therefore, the effects of upregulated CCL2 before and after PTx injection were studied in this model using T1-weighted contrast-enhanced MR imaging. As previously described, the upregulation of CCL2 within the CNS of mice causes inflammatory cells to cross the endothelial cell barrier and accumulate within the perivascular space of vessels in the brain; [17, 18] but this did not produce any clinical signs of disease. In the present study, contrast-enhanced MR images did not show any evidence of an increase in BBB permeability to a 550D Gd contrast agent in the absence of treatment with PTx. Similiarly, histological examination did not show the presence of larger 70kD dextrans in the CNS parenchyma outside vessels following intravenous administration.
There was no evidence of endothelial dysfunction in vessels surrounded by a perivascular accumulation of inflammatory cells in CCL2 transgenic mice. Staining for tight junction protein claudin-5 demonstrated no difference between CCL2 transgenic mice pre- or post-PTx injection. These observations do not rule out endothelial dysfunction at a previous point in time, as well as the possibility that extravasation occurs in a manner that does not involve disruption of tight junctions. In a previous study of mice with EAE using electron microscopy, there was no evidence of loss of integrity of the BBB during leukocyte migration . This provides supporting evidence for a transcellular migration pathway across the endothelium, with tight junctions remaining intact. Considering a paracellular migration pathway, CCL2 has been found to have a role in the alteration of tight junctions in the endothelium both in vitro and in vivo[22–24]. A decrease in expression of the tight junction proteins occludin, claudin-5, ZO-1, and ZO-2 was observed following intracerebral injection of CCL2 in mice . The effect of CCL2 on a BBB co-culture model with astrocytes was increased barrier permeability when endothelial cells were CCR2+/+ and astrocytes were CCR2−/−, but not when endothelial cells were CCR2−/− and astrocytes were CCR2+/+. These results suggest that CCL2 acts specifically on the endothelium to cause increased BBB permeability.
The transgenic mouse model used in this study is not a perfect physiologic system as it overexpresses CCL2 resulting in a 100,000 fold increase in the mRNA level of CCL2 within the brain (unpublished data by Toft-Hansen et al). Despite this limitation, the CCL2 transgenic mouse model represents a good experimental system in which leukocytes accumulate in the perivascular space, and parenchymal infiltration can be stimulated using PTx. In the present study, no signs of BBB disruption could be visualized on calculated contrast-enhanced MR maps even though inflammatory cells were found to have crossed the endothelium and accumulated in the perivascular space. The overexpression of CCL2 alone was therefore insufficient to produce BBB breakdown, defined in this study as a disruption of the endothelium and glia limitans. The accumulation of fluorescent dextrans in the perivascular space in CCL2 transgenic mice before the administration of PTx or HBSS confirms that the endothelial barrier is permeable to the tracer; however, dextrans were not found to cross the astrocytic barrier. The more confined spread of dextrans in comparison to the areas of MR enhancement suggests either greater sensitivity using MR imaging or greater diffusion of the smaller Gd-DTPA molecule. These observations from histology and contrast-enhanced MR imaging indicate that in the CCL2 transgenic mice there is increased permeability through the endothelial barrier while the astrocytic barrier remains intact and prevents the movement of inflammatory cells, 70kD dextran tracer, and 550D Gd-DTPA contrast agent into the brain parenchyma.
Wild-type mice did not show any signs of BBB disruption following PTx administration. Other studies have shown that PTx causes increased permeability to horseradish peroxidase tracer in a monolayer of brain capillary endothelial cells . Enhanced leakage across the microvasculature in mice due to PTx was also observed following histamine administration [26, 27] as well as during the onset of EAE . However, in a previous study of a SJL/J mouse model of EAE, control mice treated with PTx did not show any indications of BBB permeability .
When CCL2 transgenic mice were treated with PTx, inflammatory cells infiltrated the brain tissue surrounding blood vessels, dextran tracer leaked into the brain parenchyma, and activated microglia/macrophages were found present in brain parenchyma. The maximum area of focal enhancements on contrast-enhanced MR images was found at day 3 following PTx treatment in CCL2 transgenic mice, suggesting a transient BBB opening in this model, and the decreased contrast enhancement at day 5 indicates that the BBB disruption was beginning to resolve by this time.
Metalloproteinases degrade the extracellular matrix components of the BBB, and metalloproteinase genes were previously found to be significantly upregulated in this CCL2 transgenic mouse model following PTx administration . This occurs along with an increase in proinflammatory cytokines IL-1β and TNF-α, which play a role in leukocyte migration into the CNS . CCL2 transgenic mice showed increased levels of MMP-10, MMP-12, IL-1β and TNF-α even before PTx injection, but the results of the present study show that this is not sufficient to cause BBB permeability. The addition of PTx to the model is the additional stimulus needed to cause BBB breakdown and stimulate the cells accumulated in the perivascular space to cross the astrocytic cell barrier and enter brain parenchyma.
The CCL2 transgenic mice in our study exhibited severe disruption in the BBB that allowed a 70kD dextran tracer to move into the brain parenchyma. Permeability to a large tracer indicates that other large molecules from the blood can escape into the CNS during the inflammatory process. Large serum proteins, such as thrombin, may in turn cause local damage to brain tissue and exacerbate extracellular water accumulation [29–31]. Permeability of the BBB to large molecules in this transgenic mouse model of brain inflammation has important implications for understanding the consequences of and developing treatments for encephalomyelitis.