oxidase (HRP) in rats at the height of clinical EAE has been used. In these animals, tracer leakage was only noted in inflamed venules with diameters of.
American Journal of Pathology, Vol. 137, No. 5, November 1990 Copyright © American Association ofPathologists
Mechanisms of Edema Formation in Experimental Autoimmune Encephalomyelitis The Contribution of Inflammatory Cells
Luz Claudio, Yvonne Kress, Jason Factor, and Celia F. Brosnan From the Division of Neuropathology, Department of Pathology, Albert Einstein College of Medicine,
Bronx, New York
Most of the central nervous system (CNS) endothelium regulates the passage of solutes andfunctions as a blood-brain barrier (BBB). During experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, loss of BBB function occurs. The authors have previously shown an increase in endothelial transcytotic activity associated with decreased mitochondrial content as evidence ofBBB dysfunction in EAE. These changes occurred in the capillary bed and correlated with CNS edema and clinical signs. In the present report, a fixation procedure before infusion of the intravascular tracer horseradish peroxidase (HRP) in rats at the height of clinical EAE has been used. In these animals, tracer leakage was only noted in inflamed venules with diameters of 12 to 19 AL. The authors detected several mechanisms of passive leakage: 1) increased junctional permeability; 2) increased interendothelial space; 3) leakage alongside migrating inflammatory cells. Some small capillaries showed necrotic changes with minimal tracer leakage. This report demonstrates that BBB disruption also occurs via nonendocytic mechanisms that may be induced by inflammatory cells. (Am J Pathol 1990; 137: 1033-1045)
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) that is frequently used as a model for multiple sclerosis (MS). In susceptible species, clinical signs of paralysis have been shown to depend on the sensitization of T lymphocytes to myelin basic protein (MBP) and to correlate with the extent of perivascular cellular inflammation and edema in the CNS.1 2 Further studies
have shown that T-lymphocytes of the helper phenotype are responsible for passive transfer of the disease and that idiotypic suppressor cells and anti-ergotypic cells are generated after sensitization.3 The development of the lesion in EAE is characteristic of a delayed-type hypersensitivity (DTH) reaction in which augmenting inflammatory cells (predominantly cells of the monocyte-macrophage series) constitute a major component of the cellular infiltrate.45 Although in some reports clinical signs have been shown to depend on the involvement of these augmenting inflammatory cells,6 in other studies a role for a DTH response has been questioned.78 However, disruption of the blood-brain barrier (BBB) appears to be a critical event in the disease process.911 An analysis of the relationship between edema and cellular inflammation in EAE has suggested that the two may be dissociated, and Leibowitz and Kennedy12 have proposed that this may be due to the release of soluble lymphokines. Sensitized lymphocytes in culture release a number of factors after activation that are collectively known as cytokines. Although many of these cytokines were initially identified by their role in activation and expansion of the immune response, more recently it has become clear that they affect a whole range of activities, including the initiation of inflammation and the increase in vascular permeability.13'19 Additional evidence has suggested that endothelial cells are damaged by these cytokines. For example, the in vivo administration of interleukin-2 (IL-2) and the transfer of lymphokine-activated killer cells has been accompanied by evidence of systemwide capillary damage and vessel leakage.20 In this study, we have attempted to define the contribution of inflammatory cells to edema formation in Lewis rats sensitized to develop EAE. To this end, we have examined the mechanism of edema formation associated with cellular inflammation using horseradish peroxidase Supported by NIH grants NS 23247, NS 11920, and Training Grant NS 07098, and grant 1089 from the National MS Society. Accepted for publication June 6, 1990. Address reprint requests to Luz Claudio, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
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(HRP) as a tracer and have determined the distribution of sensitized lymphocytes in the lesion by the passive transfer of '4C-labeled cells.
Materials and Methods Sensitization Male Lewis rats (Charles River Breeding Laboratories, Wilmington, MA) weighing approximately 250 g were used. Guinea pig myelin was prepared according to the method of Norton and Poduslo21 and stored lyophilized at -20°C. Before use, myelin was resuspended at 5 mg/ml in a 1: 1 sterile solution of distilled water and 0.9% saline. The mixture was emulsified in an equal volume of complete Freund's adjuvant (CFA) containing 10 mg/ml of Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, Ml). Animals were sensitized on day 0 by injection of 0.05 ml of the inoculum into only one hind footpad or subcutaneously into the flank. Controls were injected with CFA emulsified 1:1 with 0.9% saline.
Dissection and Postfixation The cord was dissected free from the vertebrae and divided into cervical, thoracic, and lumbar segments. The tissue was postfixed for 2 hours in 3% glutaraldehyde/ 0.1 mol/l cacodylate buffer and stored overnight in cacodylate buffer containing 7% sucrose at 40C. Vibratome sections (40 u) were cut and pre-incubated with diaminobenzidine tetrahydrochloride (DAB, Sigma) at 1 mg/ml in 0.1 mol/l TRIS buffer (pH 7.4) for 1/2 hour. The sections were washed with TRIS buffer and reincubated with DAB containing 0.02% H202 for 5 minutes in the dark. For light microscopy, sections were dehydrated and mounted on glass slides. For electron microscopy, sections were posffixed in 2% osmium tetroxide, dehydrated and preembedded in epoxy resin (Epon, Polysciences, Warrington, PA) between two plastic coverslips. This allowed for proper orientation of the vibratome sections. After polymerization, they were cut and re-embedded in plastic
capsules.
Tissue Sectioning Clinical Assessment of Disease Beginning on day 7, animals were weighed and assessed for clinical signs of disease on a daily basis. A clinical index (Cl) was used to grade the animals on a scale of 1 to 5: Cl + 1, abnormal gait and flaccid tail accompanied by weight loss (more than 10 g); Cl + 2, a mild but definite weakness of one or both hind legs or severe ataxia; Cl + 3, minimal hind leg movement; Cl + 4, no hind leg movement accompanied by forelimb involvement; and Cl + 5, moribund state with impaired respiration and little or no spontaneous movement.
Tracer Infusion For the prefixation procedure, three normal, three CFAsensitized, and seven EAE rats at the height of clinical disease (Cl = 4) were used. In this procedure the animals were first anesthetized with pentobarbital and then perfused via the ascending aorta with 10 ml of 3% glutaraldehyde in 0.1 mol/l (molar) cacodylate buffer (pH 7.4). Immediately, 12,500 units of horseradish peroxidase (HRP type VI, Sigma Chemical Co., St. Louis, MO) dissolved in 10 ml 0.9% saline were slowly perfused through the left ventricle. This was followed by perfusion with 60 ml fixative solution. Two other EAE animals (Cl = 4) were injected with the same dose of HRP (12,500 units dissolved in 0.5 ml of 0.9% saline) into the tail vein 5 minutes before perfusion with fixative, as described previously.'
One-micron-thick sections were dry mounted onto glass slides and stained with toluidine blue for visualization in the light microscope. In this way, areas to be examined by electron microscopy were selected. The focus was on the area of the gray/white matter border of the lateral columns, where lesions occurred most often. Thin sections (60 nm) of the areas of interest then were cut from the corresponding blocks, collected on copper grids, and stained with uranyl acetate for 10 minutes and with lead citrate for 5 seconds.
Passive Transfer of Activated Lymphocytes EAE was induced by the passive transfer of activated lymphocytes according to established protocols.-3 In brief, lymphocytes were isolated from MBP-sensitized animals 10 days after sensitization and cultured in vitro for 72 hours in the presence of 2 ug/ml of MBP. During the last 18 hours of culture, 1 gCi of 14C-thymidine per 2 x 107 cells was added (methyl 14C-thymidine, specific activity 57 mCi/nmol, Amersham, Arlington Heights, VA) were added to the medium. The cells were washed 3X with Roswell Park Memorial Institute media, the count adjusted to 8 X 107 cells/ml, and 0.5 ml injected into the tail vein. An aliquot of the cells was smeared onto a slide and processed for autoradiography. The percentage of labeled lymphoblasts in these preparations was >80%.
Autoradiography Vibratome (40-,u) and Epon-embedded (1 -A) sections were mounted onto acid-washed slides that had been coated
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with gelatin (0.5% gelatin and 0.05% chrome alum in water). The slides were air dried at room temperature, coated with emulsion in the dark (Kodak NTB 3 emulsion [Rochester, NY] diluted 1:1 with water at 430C), air-dried, and stored desiccated in the dark for varying lengths of time. The emulsion was developed with Dektol (Kodak) (1:1 for 2 minutes at 170C, fixed in Kodak Fixer for 7 minutes, and the slides then were washed for 30 minutes in water. Vibratome sections were dehydrated through graded alcohols and mounted in Permount (Fisher Scientific, New York, NY). Epon-embedded sections were counterstained with 1% toluidine blue and mounted in Permount.
Quantitation The number and distribution of 14C-labeled cells was determined by dark-field examination of the tissue and by systematic scanning of the sections using a X 40 objective.
Results Light Microscope Studies The contribution of inflammatory cells to alterations in vascular permeability was most clearly evident in animals that had been fixed before perfusion with tracer during the onset phase of the acute clinical episode (11 to 12 dpi). In these animals, leakage of tracer into the cord parenchyma was only apparent in vessels that showed evidence of inflammation. These vessels were venules with an average diameter of 12 to 19 ,u (Figure 1 a, arrows, and 1 b). In contrast, sections from control CFA and normal animals showed no extravasation of tracer after the pre-fixation procedure (Figure 1c). The contribution of inflammatory cells to alterations in vascular permeability in EAE was most clearly evident at the electron microscopic level.
Electron Microscope Studies The results of the prefixation studies suggest that, in addition to active endocytic transfer of label, three possible mechanisms of passive transport of tracer may be present: 1) the induction of changes in the permeability of the tight junction; 2) disruption of the junctional adhesions with an increase in the interendothelial space; and 3) the diffusion of tracer alongside the migrating inflammatory cells. Indications of generalized endothelial cell damage were also found. Evidence for these changes and their relation to leakage of HRP is discussed below.
Junctional Leakage Evidence of tracer along the length of the junctional complex was observed in association with inflammatory cells (Figures 2, 3) and in areas with extensive leakage of tracer into the cord parenchyma. This evidence of junctional leakage appeared to precede actual migration of the cell and suggests that factors released by inflammatory cells may alter the nature of the tight junction. Tight junctions in the vicinity of adherent inflammatory cells show evidence of tracer throughout the junctional complex. To verify the presence of HRP throughout the junctional cleft, goniometer stage electron microscopy was used (Figure 4). Figure 5 shows two stereopair images of the same junction, confirming the presence of tracer throughout the intercellular space.
Interendothelial Space In areas of extensive inflammation, the junctional adhesions appeared disrupted, increasing the width of the interendothelial space (Figure 6). In these instances, the inflammatory cells were primarily detected in the parenchyma, suggesting that this opening of the junction resulted from the passage of inflammatory cells across the endothelial wall. Extensive and diffuse leakage of tracer into the parenchyma was present around these areas. Although it is not possible to prove that these areas represent interendothelial cell junctions, the presence of distinct cell membranes between the opposing cell surfaces would lend credence to this interpretation (Figure 6, insert). Tracer leakage along migrating inflammatory cells: Reaction product appeared entrapped along the periphery of inflammatory cells as they migrated through the endothelium (Figure 7). Tight junctions were often seen near the point of entry of inflammatory cells. In some instances, tracer was observed within these tight junctions, while in others its presence was not clear.
Endothelial Cell Damage Occasional evidence of damage to the endothelial cell was observed. In most cases, the migration of inflammatory cells appeared to cause no direct damage to the endothelial cell itself. However, in certain vessels the endothelial cell was flooded with tracer and possessed numerous vacuoles suggestive of impending necrosis. The surrounding tissue showed slight if any staining with HRP (Figure 8). These vessels were primarily capillaries.
Passive Transfer of '4C-labeled Lymphocytes To define further the role of inflammatory cells in initiating damage to endothelial cells, MBP-activated lymphocytes
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Figure 1. Vibratome sectionsfrom lumbar spinal cords of rats perfusion-fixed before the intravascular infusion of HRP. a: In animals with EAE (CI +4), tracer extravasation occurred almost exclusively in inflamed vessels of the gray-white matter border (arrows) (X32). An area of bemorrhage is also shown (curved arrow). A higher magnification of an affected vessel is shown in b (X320). c represents a comparable sectionfrom an animal sensitized with CFA alone (X32). The prefixation procedure did not affect vesselpermeability in the control.
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examined, the mean number of inflammatory cells per section was 195. The mean number of labeled cells per 1 -i section was 0.73 ± 0.8, which represented 0.37% of total inflammatory cells. The localization of these cells was 64.7% associated with vessels, 23.5% in the parenchyma, and 11.8% in the meninges. Thus, the distribution of the labeled cells was essentially the same for the vibratome and the Epon sections and showed that the majority of the passively transferred labeled cells were found in association with the vasculature. An example of an Eponembedded section is shown in Figure 9.
Discussion
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