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• W2026620460 abstract "The cellular and microvascular responses of JC Lewis rats to an intravenous injection of activated T cells specific for ovalbumin were examined with the retinal whole mount technique. The retina was examined at various times post-injection (pi) with the use of antibodies to the αβ T cell receptor (TCR) or to major histocompatibility complex class II (MHC II), the monoclonal antibody ED1, and intravascular tracers. By 12 hours pi, small numbers of TCR+, ED1+, and MHC II+ cells were present within the lumen of retinal vessels, and minor breakdown of the blood-retinal barrier (BRB) and microglial activation were evident. The intensity of these responses had increased by 1 day pi, when small numbers of TCR+ cells had also undergone extravasation. By 2 to 3 days pi, the numbers of TCR+, ED1+, and MHC II+ cells in the retinal parenchyma had increased, but the BRB breakdown and microglial activation had subsided. Thus, in the absence of target antigen, activated T cells induced limited and transient breakdown of the BRB, microglial activation, and the extravasation of ED1+, MHC II+ monocytes. In contrast, the retina of rats that received an intraocular injection of ovalbumin in addition to the intravascular injection of T cells showed massive cellular recruitment and breakdown of the BRB. These results indicate that an increase in the number of activated T cells in the circulation, such as that which occurs during viral or bacterial infection, has the potential to result in transient breakdown of the BRB and a mild local microglial response. The cellular and microvascular responses of JC Lewis rats to an intravenous injection of activated T cells specific for ovalbumin were examined with the retinal whole mount technique. The retina was examined at various times post-injection (pi) with the use of antibodies to the αβ T cell receptor (TCR) or to major histocompatibility complex class II (MHC II), the monoclonal antibody ED1, and intravascular tracers. By 12 hours pi, small numbers of TCR+, ED1+, and MHC II+ cells were present within the lumen of retinal vessels, and minor breakdown of the blood-retinal barrier (BRB) and microglial activation were evident. The intensity of these responses had increased by 1 day pi, when small numbers of TCR+ cells had also undergone extravasation. By 2 to 3 days pi, the numbers of TCR+, ED1+, and MHC II+ cells in the retinal parenchyma had increased, but the BRB breakdown and microglial activation had subsided. Thus, in the absence of target antigen, activated T cells induced limited and transient breakdown of the BRB, microglial activation, and the extravasation of ED1+, MHC II+ monocytes. In contrast, the retina of rats that received an intraocular injection of ovalbumin in addition to the intravascular injection of T cells showed massive cellular recruitment and breakdown of the BRB. These results indicate that an increase in the number of activated T cells in the circulation, such as that which occurs during viral or bacterial infection, has the potential to result in transient breakdown of the BRB and a mild local microglial response. Endothelial barriers, including the blood-brain barrier (BBB), the blood-retinal barrier (BRB), and the blood-nerve barrier (BNB), shield the nervous system from circulating agents, such as immunoglobulins, that might prove toxic. These barriers also prevent the entry of resting leukocytes from the circulation. Activated T lymphocytes, however, are able to penetrate the barriers through the action of their surface enzymes and adhesion molecules,1Hickey WF Hsu BL Kimura H T-lymphocyte entry into the central nervous system.J Neurosci Res. 1991; 28: 254-260Crossref PubMed Scopus (1007) Google Scholar, 2Wekerle H Linington C Lassmann H Meyermann R Cellular immune reactivity within the CNS.Trends Neurosci. 1986; 9: 271-277Abstract Full Text PDF Scopus (697) Google Scholar and it is generally assumed that there are no implications for vascular integrity if there is no antigen recognition in the tissue. During their surveillance of a tissue such as the central nervous system (CNS),3Hu P Pollard J Hunt N Chan-Ling T Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 487-498Crossref PubMed Scopus (21) Google Scholar if they do not encounter a relevant antigen presented appropriately by an antigen-presenting cell, activated T cells return to the circulation or die by apoptosis.4Hickey WF Lassmann H Cross AH Lymphocyte entry and the initiation of inflammation in the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, New York1997: 200-225Google Scholar, 5Miller DH Rudge P Johnson G Kendall BE Macmanus DG Moseley IF Barnes D McDonald W Serial gadolinium enhanced magnetic resonance imaging in multiple sclerosis.Brain. 1988; 111: 927-939Crossref PubMed Scopus (364) Google Scholar Magnetic resonance imaging of individuals with multiple sclerosis (MS), a relatively common inflammatory demyelinating disease of the CNS, has revealed that breakdown of the BBB is the earliest demonstrable abnormality in the formation of new lesions and in the extension of old lesions.5Miller DH Rudge P Johnson G Kendall BE Macmanus DG Moseley IF Barnes D McDonald W Serial gadolinium enhanced magnetic resonance imaging in multiple sclerosis.Brain. 1988; 111: 927-939Crossref PubMed Scopus (364) Google Scholar Given that this breakdown of the BBB is thought to play a fundamental role in the pathogenesis of MS,6Poser CM The pathogenesis of multiple sclerosis: additional considerations.J Neurol Sci. 1993; 115: S3-S15Abstract Full Text PDF PubMed Scopus (58) Google Scholar it is important to understand the mechanism by which it occurs. Breakdown of the BBB is always associated with cellular infiltration in individuals with MS.7Lassmann H Suchanek G Ozawa K Histopathology and the blood-cerebrospinal fluid barrier in multiple sclerosis.Ann Neurol. 1994; 36: S42-S46Crossref PubMed Scopus (118) Google Scholar In rats with experimental allergic encephalomyelitis (EAE), an experimental model of MS, activated T cells specific for neural antigens such as myelin basic protein (MBP) or the S100 protein accumulate within the CNS and induce breakdown of the BBB.8Linington C Bradl M Lassmann H Brunner C Vass K Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/oligodendrocyte glycoprotein.Am J Pathol. 1988; 130: 443-454PubMed Google Scholar, 9Wekerle H Kojima K Lannes VJ Lassmann H Linington C Animal models.Ann Neurol. 1994; 36: S47-S53Crossref PubMed Scopus (336) Google Scholar However, there is no substantial evidence that MBP or any other neural component is a major autoantigen in MS. The demonstration of an association between MS attacks and viral infections10Andersen O Lygner P Bergstrom T Andersson M Vahlne A Viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study.J Neurol. 1993; 240: 417-422Crossref PubMed Scopus (222) Google Scholar, 11Sibley W Bamford C Clark K Clinical viral infections and multiple sclerosis.Lancet. 1985; 1: 1313-1315Abstract PubMed Scopus (508) Google Scholar suggests that T cells reactive to nonneural antigens, such as those associated with viruses, also might induce CNS inflammation. Indeed, we have previously shown that activated T cells specific for the nonneural antigen ovalbumin (OVA. are able to induce breakdown of the BNB.12Pollard JD Westland KW Harvey GK Jung S Bonner J Spies JM Toyka KV Hartung HP Activated T cells of nonneural specificity open the blood-nerve barrier to circulating antibody.Ann Neurol. 1995; 37: 467-475Crossref PubMed Scopus (74) Google Scholar The retina is an ideal tissue in which to characterize the microvascular and cellular responses of the CNS to an intravascular injection of activated T cells of nonneural specificity, because it is possible to visualize the entire retinal vascular plexus with the normal relations among the glial, vascular, and neuronal elements intact.13Chan-Ling T Neill AL Hunt NH Early microvascular changes in murine cerebral malaria detected in retinal wholemounts.Am J Pathol. 1992; 140: 1121-1130PubMed Google Scholar, 14Ma N Hunt NH Madigan MC Chan-Ling T Correlation between enhanced vascular permeability, up-regulation of cellular adhesion molecules and monocyte adhesion to the endothelium in the retina during the development of fatal murine cerebral malaria.Am J Pathol. 1996; 149: 1745-1762PubMed Google Scholar, 15Medana IM Hunt NH Chaudhri G Tumor necrosis factor-α expression in the brain during fatal murine cerebral malaria: evidence for production by microglia and astrocytes.Am J Pathol. 1997; 150: 1473-1486PubMed Google Scholar In particular, with the use of intravascular barrier tracers and cell-specific reagents, it is possible to colocalize sites of cellular accumulation with sites of breakdown of the BRB. The retinal whole mount technique has the additional advantage that arteries, capillaries, and venules are readily identified, thereby allowing accurate localization of specific cellular and vascular changes to specific regions of the CNS microvasculature. Our previous application of this technique resulted in the detection of BRB breakdown and the identification of small numbers of inflammatory infiltrates in the retinas of rats with EAE3Hu P Pollard J Hunt N Chan-Ling T Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 487-498Crossref PubMed Scopus (21) Google Scholar and of mice with experimental cerebral malaria.13Chan-Ling T Neill AL Hunt NH Early microvascular changes in murine cerebral malaria detected in retinal wholemounts.Am J Pathol. 1992; 140: 1121-1130PubMed Google Scholar We have now characterized the cellular and microvascular responses, in the absence or presence of target antigen, to an intravenous injection of activated T cells specific for OVA. Because the barrier properties of retinal vessels are similar to those of vessels elsewhere in the CNS,16Chan-Ling T Glial, neuronal and vascular interactions in the mammalian retina.Prog Retinal Res. 1994; 13: 357-389Crossref Scopus (54) Google Scholar the changes observed in the present study are highly relevant to those characteristic of MS and other inflammatory CNS disorders. A total of 64 adult male JC Lewis rats aged 10 to 14 weeks were used in this study. Twelve animals were used as naive controls. The first experimental group of 12 animals received only an intravenous injection of 5 × 106 activated OVA-specific T cells (GH2 T cell line) in 0.9 ml of RPMI medium, and they were examined 12 hours and 1, 2, and 3 days postinjection (pi). The second group of 40 animals received both the intravenous injection of activated OVA-specific T cells and intraocular injections of OVA and vehicle (see below) within 5 minutes of each other. Animals in this group were examined at 6 and 12 hours and 1, 2, 3, 5, and 7 days pi. Anesthesia for intravenous injections and terminal experiments were as previously described.17Hu P Pollard J Hunt N Taylor J Chan-Ling T Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 475-486Crossref PubMed Scopus (37) Google Scholar Twenty to thirty microliters of an OVA (Sigma, St. Louis, MO. solution in RPMI (1 mg/ml; sterilized by passage through a low-protein binding filter) were injected at a single site in the region of the left retina with the use of a Hamilton-type syringe (SGE Scientific, Melbourne, Australia) with a Luer lock fitting (N09-S2397) for attachment to disposable 31-gauge needles. The needle was introduced into the globe at a site immediately posterior to the ora serrata and the injection was performed under direct visual guidance through a dissecting microscope, with the aim of depositing the solution adjacent to the retina. The right eye received an identical injection with RPMI alone. OVA-reactive T cells (GH-2 T cell line) were prepared as previously described.12Pollard JD Westland KW Harvey GK Jung S Bonner J Spies JM Toyka KV Hartung HP Activated T cells of nonneural specificity open the blood-nerve barrier to circulating antibody.Ann Neurol. 1995; 37: 467-475Crossref PubMed Scopus (74) Google Scholar Retinal whole mounts18Chan-Ling T Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.Microsc Res Techniq. 1997; 36: 1-16Crossref PubMed Scopus (97) Google Scholar and HRP histochemistry,17Hu P Pollard J Hunt N Taylor J Chan-Ling T Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 475-486Crossref PubMed Scopus (37) Google Scholar, 19Chan-Ling T Tout S Hollander H Stone J Vascular changes and their mechanisms in the feline model of retinopathy of prematurity.Invest Ophthalmol Vis Sci. 1992; 33: 2128-2147PubMed Google Scholar were prepared as previously described. The molecular size of HRP (40 kd) is similar to those of serum proteins, and this similarity has been taken advantage of in a widely used method for detection of BBB breakdown.20Stewart PA Farrell CR Farrell CL Hayakawa E Horseradish peroxidase retention and washout in blood-brain barrier lesions.J Neurosci Meth. 1992; 41: 75-84Crossref PubMed Scopus (23) Google Scholar The Griffonia simplicifolia isolectin B4 (GS lectin), which labels α-D galactose residues, was used to visualize microglia and macrophages. GS lectin histochemistry was performed as previously described.21Chan-Ling T Halasz P Stone J Development of retinal vasculature in the cat: stages, topography and mechanisms.Curr Eye Res. 1990; 9: 459-478Crossref PubMed Scopus (105) Google Scholar Immunohistochemistry for ED1, TCR, and MHC II was performed as previously described.3Hu P Pollard J Hunt N Chan-Ling T Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 487-498Crossref PubMed Scopus (21) Google Scholar, 17Hu P Pollard J Hunt N Taylor J Chan-Ling T Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 475-486Crossref PubMed Scopus (37) Google Scholar For ED1, αβTCR, and MHC II immunohistochemistry, each retina was divided equally at the optic disk into three sectors and one of the above markers was applied to each sector. Cell density was determined for each marker by counting immunoreactive cells using a 10× or 20× objective at intervals of 1 mm2 and averaged for the entire sector. Retinas were examined after intravascular injection of Evans blue, bisbenzimide, and Monastral blue as previously described.3Hu P Pollard J Hunt N Chan-Ling T Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 487-498Crossref PubMed Scopus (21) Google Scholar, 13Chan-Ling T Neill AL Hunt NH Early microvascular changes in murine cerebral malaria detected in retinal wholemounts.Am J Pathol. 1992; 140: 1121-1130PubMed Google Scholar Evans blue (Sigma) is an acid dye of the diazo group that binds to albumin in the blood, allowing sites of barrier breakdown to be readily detected. Bisbenzimide (or Hoechst stain, H33258; Calbiochem-Novabiochem, San Diego, CA) interacts with DNA and stains nuclei of all cell types. When the BRB is intact, retinal exposure to intravenously administered bisbenzimide is restricted to the vascular endothelial cells that line the lumen of retinal vessels. However, if the BRB is disrupted, the dye can access the retinal parenchyma, with the result that all cells, including the somas of neurons and glia, are labeled. Bisbenzimide also labels the nuclei of inflammatory cells that accumulate and adhere to the vessel wall during the progression of disease. Monastral blue is a colloidal dye, the particles of which coat, or are ingested by, activated monocytes.22Neill AL Hunt NH Pathology of fatal and resolving cerebral malaria in mice.Parasitology. 1992; 105: 165-175Crossref PubMed Scopus (123) Google Scholar In animals that received only an intravenous injection of OVA-specific activated T cells, few TCR+ lymphocytes were apparent within the lumen of retinal vessels at 12 hours pi (Figure 1A), although the number was consistently higher than that apparent for naive controls; counts across the entire retina at this time point yielded values of 1.77 ± 1.29 and 0.15 ± 0.15 TCR+ cells/mm2 for injected and naive control animals, respectively (Table 1). At this time, no TCR+ cells were detected in the parenchyma of the retina (Table 1). By days 1 to 2 pi, whereas the number of TCR+ cells within the lumen of vessels remained unchanged, small numbers of such cells had undergone extravasation and were apparent within the retinal parenchyma (Figure 1B). The number of intravascular TCR+ cells also remained unchanged at days 3 to 5 pi; in contrast, the number of TCR+ cells in the parenchyma had increased, and was significantly greater than that for naive controls from day 2 pi onward (Table 1). However, even at days 3 to 5 pi, the number of these cells in the parenchyma of injected rats was still low, 0.2 ± 0.1 cells/mm2, and they remained within ∼20 μm of the nearest vessel.Table 1Density of TCR+, ED1+, and MHC II+Cells within the Lumen of Retinal Vessels and the Retinal Parenchyma of JC Lewis Rats after Intravascular Injection of OVA-Activated T CellsTCR+ cell density (cells/mm2)ED1+ cell density (cells/mm2)MHC II+ cell density (cells/mm2)ParenchymaParenchymaTime (pi)nIntravascularParenchymaIntravascularMicroglial-likeMonocyte-likeIntravascularMicroglial-likeMonocyte-likeNaive controls30.15 ± 0.150.00 ± 0.000.20 ± 0.200.00 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.000.00 ± 0.0012 hr31.77 ± 1.290.00 ± 0.001.03 ± 0.752.33 ± 1.35*0.01 ± 0.020.83 ± 0.49*0.00 ± 0.000.00 ± 0.00Day 131.60 ± 1.220.04 ± 0.071.33 ± 0.50*3.60 ± 3.140.00 ± 0.000.64 ± 0.830.00 ± 0.000.00 ± 0.00Day 231.70 ± 1.130.07 ± 0.03*1.87 ± 1.201.97 ± 2.000.41 ± 0.521.81 ± 1.13*0.00 ± 0.000.12 ± 0.62Day 331.40 ± 0.40*0.20 ± 0.10*0.67 ± 0.470.91 ± 0.10*0.10 ± 0.00*1.24 ± 0.21*0.00 ± 0.000.11 ± 0.10Data are means ± SD. *P < 0.05 vs. control rats (Student's one-tailed t-test). pi, postinjection. Open table in a new tab Data are means ± SD. *P < 0.05 vs. control rats (Student's one-tailed t-test). pi, postinjection. A small increase in the number of ED1+ (Figure 1C) and MHC II+ cells (Figure 1E) within the lumen of retinal vessels were apparent in injected animals at 12 hours pi. Cell counts for ED1+ monocytes yielded values of 1.03 ± 0.75 and 0.2 ± 0.2 cells/mm2 for injected and naive control animals, respectively. Cell counts for MHC II+ cells in naive controls were 0 ± 0 compared to 0.83 ± 0.49 cells/mm2 at 12 hours pi (Table 1).3Hu P Pollard J Hunt N Chan-Ling T Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 487-498Crossref PubMed Scopus (21) Google Scholar The number of ED1+ and MHC II+ cells within the lumen of vessels of injected animals increased slightly on days 1 and 2 pi but thereafter decreased (Table 1). From day 2 to day 3 pi, only small numbers of ED1+ (Figure 1D) and MHC II+ cells (Figure 1F) had undergone extravasation and were evident within the retinal parenchyma of injected rats. The BRB is intact in naive control adult JC Lewis rats (Figure 2A). Thus, intravascular injection of HRP and the modified trichrome technique demonstrated that the retinal vessels exhibited a sharp outline and retained tracer within the vessel lumen, with no detectable tracer leakage into the tissue parenchyma. Intravenous injection of OVA-activated T cells resulted in a limited breakdown of the BRB even at 12 hours pi, as was evident by increased HRP accumulation at junctions between endothelial cells and by a slight blurring of vessel outlines (Figure 2C). This leakiness was increased slightly at 24 hours pi using both HRP (Figure 2E) and the modified trichrome technique (Figure 3, A −C). At day 2 pi, uneven accumulation of HRP on the venous vessel wall was evident, but no leakage of HRP was observed (Figure 2G).Figure 3A−C: Examination by the modified trichrome technique at day 1 pi of the same venous segment in the retina of a rat that received only an intravascular injection of OVA-activated T cells. A limited breakdown of the BRB is evident as a weak halo of Evans blue (A) and Hoechst stain (B. surrounding the vessel outline. Adherent Monastral blue-positive monocytes (arrows) are apparent by Hoechst staining (B) and transmitted light microscopy (C). D−J: Difference in the microvascular and cellular responses between venous and arterial segments in OVA-injected eyes after an intravenous injection of OVA-activated T cells. D−F: Despite being in close proximity, the arteries exhibited no leakage of Evans blue, Hoechst stain, or Monastral blue, whereas the venous segments showed a marked breakdown of the BRB, which colocalized with sites of accumulation of Monastral blue-positive monocytes and other leukocytes at day 3 pi. Small arrows in F show the locations of adherent leukocytes. At 12 hours pi, the early activation of GS lectin-stained microglia (G, arrowheads) occurred adjacent to either arteries or veins. In contrast, adhesion and extravasation of leukocytes including, GS lectin-stained (G), ED1+ (H), MHC II+ (I), and TCR+ cells (J) were apparent only in the venous vasculature. Small arrows in H–J indicate the outlines of arteries (a) and venous segments (v). Scale bar, 50 μm (D−F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to causing a mild breakdown of the BRB, injection of OVA-activated T cells induced a limited activation of microglia, as indicated by changes in microglial morphology and up-regulation of αD galactose23Maddox DE Shibata S Goldstein IJ Stimulated macrophages express a new glycoprotein receptor reactive with Griffonia simplicifolia I-B4 isolectin.Proc Natl Acad Sci USA. 1982; 79: 166-170Crossref PubMed Scopus (117) Google Scholar and ED1 expression by resident microglia. Unlike the situation in control rats, in which microglia are stained only weakly with GS lectin and exhibit a ramified morphology3Hu P Pollard J Hunt N Chan-Ling T Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE).Brain Pathol. 1998; 8: 487-498Crossref PubMed Scopus (21) Google Scholar (Figure 2B), resident microglia became intensely labeled with GS lectin, adopted an ameboid morphology, and exhibited prominent distensions along their processes between 12 and 24 hours after injection of OVA-activated T cells (Figure 2, D and H). Although the severity of this peaked at day 1, the microglia still showed distensions along their processes at day 2 pi (Figure 2H). Topographical quantitative analysis of these microglial changes showed that by 12 hours pi, the number of ED1+ microglia-like cells in the retinal parenchyma of injected rats was 2.33 ± 1.35 cells/mm2 (Table 1); such cells were absent from control retinas. The number of these cells peaked at day 1 pi (3.60 ± 3.14 cells/mm2) and decreased gradually thereafter (Table 1). Previous investigators have reported the weakening in the intensity and reliability of GS lectin labeling in adult rat retina, making it difficult to quantify microglial numbers in naive control animals.24Ashwell KWS Hollander H Streit W Stone J The appearance and distribution of micoglia in the developing retina of the rat.Vis Neurosci. 1989; 2: 437-448Crossref PubMed Scopus (127) Google Scholar However, despite these difficulties, quantification of GS lectin-positive microglia showed a marked increase in these cells from 12 hours through to day 2 pi. GS lectin-positive microglia had an approximate density of 5 cells/mm2 in naive controls. By 12 hours pi, this had increased to approximately 15 cells/mm2, peaking at 49 cells/mm2 at day 1 pi. By day 2 pi, GS lectin-positive microglial density was approximately 44 cells/mm2; ED1+ microglia also persisted in the parenchyma at this time (Table 1). However, in the absence of target antigen, no MHC II+ microglia was found throughout the observation period (Table 1). The second group of animals received both an intravascular injection of OVA-activated T cells and intraocular injections of OVA (left eye) and RPMI (right eye, data not shown). Thus, the difference in responses between the two eyes can be attributed to the absence or presence of the target antigen. In the OVA-injected eyes, TCR+ cells were first detected within the lumen of retinal vessels at 6 hours pi (data not shown). By 12 hours pi, a substantial number (74 ± 48 cells/mm2) of TCR+ cells had undergone extravasation in the vicinity of veins (Figure 4A). From day 1 pi onward, TCR+ cells were no longer obviously aligned along retinal veins but were distributed randomly throughout the parenchyma (Figure 4B). The density of extravasated TCR+ cells was 200 ± 288 cells/mm2 at day 2pi. By 6 hours pi, substantial numbers of ED1+ monocytes were evident within the lumen of retinal veins in the OVA-injected eyes (data not shown). Large numbers of extravasated ED1+ cells (311 ± 89 cells/mm2) were first evident adjacent to retinal veins at 12 hours pi (Fig. C). Process-bearing cells in the parenchyma, likely to be resident microglia, also stained with ED1 at 12 hours pi (17 ± 20 cells/mm2. Figure 4C). The number of ED1+ monocytes in the parenchyma peaked between 12 hours and 1 day pi (data not shown). Between day 1 and day 3 pi, both ED1+ monocytic infiltrates and ED1+ microglia were apparent throughout the entire retina (Figure 4D). The monocytic nature of a significant number of ED1+ cells was confirmed by the coating of these cells by Monastral blue (Figure 4, C and D). In the OVA-injected eyes, MHC II+ cells were first detected within the lumen of retinal vessels at 6 hours pi (data not shown). By 12 hours pi, a substantial number (97 ± 67 cells/mm2) of MHC II+ cells had undergone extravasation in the vicinity of veins (Figure 4E). Process-bearing cells in the parenchyma, likely to be resident microglia, also stained with MHC II at 12 hours pi (4 ± 6 cells/mm2; Figure 4E). The number of MHC II+ monocytes in the parenchyma peaked at day 1, whereas the number of MHC II+ microglia was maximal between days 3 and 7 pi (data not shown). By day 2 pi, MHC II+ cells were no longer obviously aligned along retinal veins but were distributed randomly throughout the parenchyma (Figure 4F). Microglial activation, as revealed by the adoption of an amoeboid morphology and an increased staining with GS lectin, was apparent throughout the observation period. Resident microglia adopted an amoeboid morphology and displayed a marked increase in reactivity for GS lectin shown from 12 hours (Figure 3G) through to day 1 (Figure 4I). A substantial number of GS lectin-stained monocyte-like cells had undergone extravasation in the OVA-injected eyes by day 2 pi (Figure 4J). In the presence of the specific T cell antigen, a marked breakdown of the BRB was evident from day 1 and persisted throughout the observation period. Leakage of HRP from the vessel lumen resulted in a pronounced blurring of the vessel outline shown here for day 1 pi (Figure 4G). At day 2 pi, the extent of BRB breakdown was so great that virtually all of the enzyme had been lost from the vessel lumen and had diffused over a large region of the parenchyma (Figure 4H). Hemorrhaging was observed using the colloidal intravascular tracer Monastral blue from day 1 pi persisting until day 7 pi. Figure 2, D −F, shows marked leakage of Evans blue, Hoechst stain, and Monastral blue colocalized to a venous segment with numerous adherent leukocytes in the presence of target antigen at day 3 pi. Irrespective of the technique used to examine the extent of T cell and monocyte accumulation and extravasation and the breakdown of the BRB, the severity of the responses was consistently greater in venous segments than in arteries (Figure 3, D-J). Given the soluble nature of OVA, it would be expected that the target antigen would be equally accessible in the region of arteries and in that of veins. Figure 3, D −F, shows the same arterial and venous segments in an OVA-injected eye at 3 days pi examined under three different types of illumination according to the modified trichrome technique. The arterial segment, clearly evident from the characteristic elongated morphology of the endothelial cell nuclei, had retained the Evans blue as shown by the distinct vessel outline (Figure 3, D and E). In contrast, the venous segment exhibits a marked breakdown of the BRB, with the loss of most of the Evans blue-bound plasma albumin from the vessel lumen (Figure 3D). Accumulation of leukocytes is also greatly enhanced in the venous segment as evidenced by the large number of nuclei labeled with Hoechst stain within the lumen. Several of these adherent leukocytes were either coated by Monastral blue or had internalized the dye, indicative of their monocytic nature (Figure 3, D and E). The adherent leukocytes also had a marked effect on blood rheology, as was apparent under transmitted illumination (Figure 3F). The pronounced leakiness of the venous segment is also evident from the accumulation of Monastral blue particles in the parenchyma surrounding the vein; such particles were not detected in the parenchyma" @default.
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• W2026620460 title "Breakdown of the Blood-Retinal Barrier Induced by Activated T Cells of Nonneural Specificity" @default.
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