key: cord-1016081-4aadb0py
authors: Vinores, Stanley A; Wang, Yun; Vinores, Melissa A; Derevjanik, Nancy L; Shi, Albert; Klein, Diane A; Detrick, Barbara; Hooks, John J
title: Blood–retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and VEGF in sensitive and resistant strains
date: 2001-10-01
journal: J Neuroimmunol
DOI: 10.1016/s0165-5728(01)00374-5
sha: 2411fccd02e8fc76e3b67f918719e1d39b12c0c9
doc_id: 1016081
cord_uid: 4aadb0py

Intraocular coronavirus inoculation results in a biphasic retinal disease in susceptible mice (BALB/c) characterized by an acute inflammatory response, followed by retinal degeneration associated with autoimmune reactivity. Resistant mice (CD-1), when similarly inoculated, only develop the early phase of the disease. Blood–retinal barrier (BRB) breakdown occurs in the early phase in both strains, coincident with the onset of inflammation. As the inflammation subsides, the extent of retinal vascular leakage is decreased, indicating that BRB breakdown in experimental coronavirus retinopathy (ECOR) is primarily due to inflammation rather than to retinal cell destruction. Vascular endothelial growth factor (VEGF) is upregulated only in susceptible mice during the secondary (retinal degeneration) phase.

Ž . The neurotropic strain JHM of the mouse hepatitis Ž . virus MHV is a murine coronavirus that induces a biphasic retinal disease when injected intraocularly into BALBrc Ž . mice Robbins et al., 1990 Robbins et al., , 1991 . The early phase of the disease occurs from 1 to 7 days post-inoculation and involves a retinal vasculitis associated with the presence of infectious virus. The late phase of the disease begins at about day 10 post-inoculation and involves retinal degeneration associated with the presence of autoantibodies directed against the neuroretina and the retinal pigmented Ž . epithelium RPE . Infectious virus, viral antigens, and inflammatory cells are absent during the late phase and virus-neutralizing antibodies are produced during both phases. When CD-1 mice are similarly inoculated, only the Ž . early phase of the disease is induced Wang et al., 1996 ; antiviral antibodies are produced, but anti-retinal autoanti-Ž . bodies are not Hooks et al., 1993 . Ž . Vascular endothelial growth factor VEGF is well characterized as an angiogenic agent and a vascular perme-Ž ability factor Senger et al., 1983 Senger et al., , 1986 Roberts and . Palade, 1995; Dvorak et al., 1995; Ozaki et al., 1997 , but recent findings show that it is also involved with the recruitment and activation of inflammatory cells and their Ž adhesion to the vascular endothelium Barleon et al., 1996;  . Clauss et al., 1996; Melder et al., 1996; Lu et al., 1999 . A marked upregulation of VEGF occurs in the inner retinas of rats developing experimental autoimmune uveoretinitis Ž . Ž . EAU Luna et al., 1997; Vinores et al., 1998a . In these rats, there is inflammatory cell infiltration and an autoim-Ž . mune reaction with blood-retinal barrier BRB breakdown, but no angiogenesis. Experimental coronavirus Ž . retinopathy ECOR of murine retinas provides a model with inflammatory cell infiltration in the early phase and an autoimmune reaction in the late phase, when BALBrc mice are used. In CD-1 mice, the autoimmune response is 0165-5728r01r$ -see front matter q 2001 Elsevier Science B.V. All rights reserved.

Ž . PII: S 0 1 6 5 -5 7 2 8 0 1 0 0 3 7 4 -5 ( ) absent; therefore, differences that may be observed in BRB breakdown and the presence andror distribution of VEGF and its receptors, when comparing these two strains, are likely to relate to the development of retinal degenerative disease. This model will be used to determine at what phase BRB breakdown occurs, how extensive it is, and whether it is reversible. The immunohistochemical localization of extravasated albumin is an established method for visualizing the location and extent of sites of BRB Ž breakdown Vinores, 1995; Vinores et al., 1989 Vinores et al., , 1990b Vinores et al., , . 1994 Vinores et al., , 1995a and it will be used to assess the integrity of the BRB. This model can also provide information as to whether VEGF may play a role in one or both of these phases. Hooks et al., 1993 . Control mice received no treatment.

At time points ranging from 1 to 62 days post virus-inoculation, a total of 218 BALBrc and 58 CD-1 mice were anesthetized and sacrificed by decapitation. The mice are handled according to the ARVO Resolution on the Use of Animals in Research. Eyes are promptly fixed in 10% buffered formalin and embedded in paraffin. Immunohistochemistry is performed on tissue sections as previously Ž described Wang et al., 1993 Wang et al., , 2000 Vinores et al., 1995a Vinores et al., ,b, . 1998a Chen et al., 1997 using 

In BALBrc mice, BRB breakdown, assessed by the immunolocalization of extravascular albumin, was not demonstrated in uninjected or mock-injected eyes or in JHM-injected eyes for the first 5 days after inoculation of Ž . coronavirus Table 1 , Fig. 1A . By 6 days after inoculation, focal sites of retinal vascular leakage were evident Ž . Inflammation was demonstrated by immunohistochemical staining for Mac-1, a marker for monocytes and natural Ž . killer cells Ault and Springer, 1981 

The number of eyes showing BRB breakdownrtotal number of eyes.

) Ž . The difference between strains is statistically significant p s 0.002 based on Fisher's Exact Test. Ž . BALBrc mouse, immunostaining for albumin brown was confined to the vessels arrowheads . In the choroid bottom , diffuse staining was evident Ž . because the choroidal vessels are fenestrated and do not have a blood-tissue barrier function. B In a BALBrc mouse, 6 days after intraocular injection of coronavirus, leakage is seen emanating from a retinal vessel. The extravasated albumin is visualized as a perivascular diffuse brown reaction product Ž . Ž . arrow . C In a BALBrc mouse, 62 days after intraocular injection of coronavirus, immunoreactive albumin is seen permeating the RPE and entering the Ž . Ž. retinal parenchyma between arrows . This represents a breach of the outer BRB. D In a CD-1 mouse, 1 day after intraocular injection of coronavirus, the Ž .Ž. BRB is intact with immunoreactivity within the retina limited to the vessels arrowheads . E In a CD-1 mouse, 8 days after intraocular injection of Ž . coronavirus, widespread, diffuse albumin positivity is evident throughout the retina. F In a CD-1 mouse, 21 days after intraocular coronavirus inoculation, albumin reactivity within the retina is confined to the vessels. retinal vascular leakage continued, but widespread BRB breakdown was not seen. inoculation, but did not develop retinal degenerative disease as do the BALBrc mice, also did not show any BRB Ž abnormalities for the first 4 days post-inoculation Table 1 ;

. Fig. 1D . At 6-7 days post-inoculation, all eyes showed moderate BRB dysfunction. The extent of BRB breakdown increased in CD-1 mice to a peak at 8 days post-inocula-Ž . tion Fig. 1E , when three of five eyes showed severe BRB breakdown and the remaining two showed moderate breakdown. Moderate or severe lymphocyte infiltration was demonstrated in 9 of 10 coronavirus-infected CD-1 mouse Ž . retinas during the acute phase days 6-8 of coronavirus Ž . infection Figs. 2E,F and 4 , which was accompanied by Ž . moderate or severe BRB breakdown in all animals Fig. 5 . By 10 days after virus inoculation, the inflammation had subsided and the integrity of the BRB was restored. The retinal architecture appeared normal and infiltrating cells were not observed. Some focal staining for extravascular albumin was demonstrated in two of eight eyes at 20-21 days post-inoculation, one showed staining in the outer retina and one in the inner nuclear layer, but in the Ž . remaining eyes, the BRB was intact Fig. 1F . BRB breakdown was not seen in BALBrc mice that were uninjected or received mock injections of saline. Focal positivity for extravascular albumin was seen in 1 of 11 untreated CD-1 mice and 1 of 13 CD-1 mice receiving mock saline injections.

VEGF staining in the retinas of coronavirus-injected BALBrc mice was negative or weak and diffuse throughout the retina and did not differ from control or mock-injected animals from 1 to 8 days after virus inoculation. At 10 days after virus inoculation, focally intense staining for VEGF was demonstrated in the outer segments of the photoreceptors in one of two mice. By 20 days, focally intense areas of VEGF positivity were evident in the nerve fiber layer, outer plexiform layer, outer nuclear layer, and Ž . the outer segments of the photoreceptors Fig. 6A . In control BALBrc mice, VEGF staining was limited to the inner retinal surface and Muller cell processes with somë Ž . weak staining in the outer plexiform layer Fig. 6B . In CD-1 mice, constitutive staining for VEGF was somewhat stronger than in BALBrc mice, but except for one of two animals that showed more intense areas of positivity in the retina and RPE 10 days after virus inoculation, the virustreated animals did not show an increase in VEGF staining Ž . compared to the controls Fig. 6C ,D .

In BALBrc mice, staining for the VEGF receptor, flk-1, was negative in the retina and RPE in normal and virus-treated animals. Immunoreactivity for the other isotype of VEGF receptor, flt-1, was either absent or very weakly demonstrated in the inner plexiform layer and in the outer segments of the photoreceptors, but there was no correlation with the course of viral infection and no differ- . Ž . inflammatory cells , and severe ) 200 inflammatory cells . When com-Ž paring strains, the distribution shift was statistically significant ps . Ž 0.016 based on the Cochran-Armitage test for trend Agresti, 1990 ;

. Margolin, 1988 . . intense staining throughout large areas of the retina . When comparing Ž . strains, the distribution shift was statistically significant ps 0.035 Ž based on the Cochran-Armitage test for trend Agresti, 1990; Margolin, . 1988 . ence between experimental animals and controls. In addition, staining for TGFb2 was weak or absent in the retinas of BALBrc and CD-1 mice with no differences relating to the course of the virus infection or to the presence or absence of virus.

The immunolocalization of extravascular albumin has previously been shown to be a reliable means of localizing sites of BRB breakdown, at both the light and electron microscopic level, and it provides insight into the mecha-Ž nisms of BRB dysfunction in ocular diseases Vinores et al., 1989 Vinores et al., , 1990a Vinores et al., ,b, 1992 Vinores et al., , 1993a Vinores et al., ,b, 1994 Vinores et al., , 1995a Vinores et al., ,b, 1998b  . Luna et al., 1997 . In coronavirus infected BALBrc mouse retinas, the first evidence of vascular leakage occurred 6 days after the inoculation of virus and corresponded to the onset of inflammatory cell infiltration as demonstrated by Mac-1 staining. This is also the time when apoptosis is Ž . occurring within the retina Wang et al., 2000 . In CD-1 mice, BRB breakdown also coincided with inflammation and both BRB dysfunction and leukocyte infiltration peaked Ž . during the acute phase of the infection days 6-8 . Albumin extravasation during the acute phase was greater in CD-1 mice than in BALBrc mice, which correlated with a greater extent of leukocyte infiltration in CD-1 mice during this phase. Following the acute phase of infection, there was a decrease in inflammatory cells and virus ( ) Ž . Ž . Fig. 6 . VEGF immunoreactivity in ECOR. A Widespread, diffuse positivity for VEGF brown is demonstrated throughout the retina of a BALBrc Ž . Ž . Ž . mouse, 20 days after intraocular coronavirus inoculation. Immunoreactivity is stronger in the outer retina bottom . B VEGF immunoreactivity red in a Ž . Ž . 20-day control BALBrc mouse is evident along the inner retinal surface top and in Muller cell processes arrowheads . Weak positivity is also seen in Ž ) . Ž . Ž . the outer plexiform layer . C VEGF staining is absent in a CD-1 mouse retina 20 days after intraocular coronavirus inoculation. D Control CD-1 Ž . mice show a staining pattern for VEGF that is similar to that observed in normal BALBrc mice see B with positivity along the inner retinal surface and in Muller cell processes.

particles and the integrity of the BRB was restored. BRB breakdown that occurred in the late phase of the infection in BALBrc mice generally corresponded to structural damage related to retinal degenerative disease.

In rats developing EAU, the marked upregulation of VEGF in the inner retina suggests that it plays a role in the Ž pathogenesis of the disorder Luna et al., 1997; Vinores et . al., 1997b Vinores et . al., , 1998a . The specific function of VEGF in the progression of EAU is unclear; however, the autoimmune disorder involves BRB breakdown and inflammatory cell infiltration, both of which can be promoted by VEGF. Likewise, with experimental herpesvirus retinopathy, which presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis associated with virus transmission to the Ž . opposite eye Whittum et al., 1984 , VEGF is upregulated Ž . in both eyes Vinores et al., 1997a . In the herpesvirus-injected eye, VEGF upregulation coincides with inflammation, with its levels diminishing as the inflammation subsides. In the contralateral eye, VEGF induction coincides with the appearance of virus particles and the onset of inflammatory cell infiltration. In experimental coronavirus infection, VEGF upregulation did not appear to coincide with inflammatory cell infiltration andror the presence of infective virus and no differences were noted in the extent or distribution of VEGF receptors through the course of the disease; therefore, VEGF may not play a significant role in the pathogenesis of experimental coronavirus retinopathy as it does in EAU and experimental herpesvirus retinopathy. The only significant upregulation of VEGF appears late in the course of the disease, when retinal degeneration is occurring, and may result from hypoxia related to the retinal degeneration, since VEGF is Ž known to be induced under hypoxic conditions Shweiki et al., 1992; Plate et al., 1992; Goldberg and Schneider, 1994; Hashimoto et al., 1994; Minchenko et al., 1994a,b; Levy et al., 1995; Pierce et al., 1995; Vinores et al., . 1997b .

It is possible that VEGF upregulation may play a role in the retinal degeneration that occurs in the late phase of ECOR in BALBrc mice. The mechanism for this is not clear, but retinal degeneration also occurs in a line of transgenic mice in which VEGF is produced in the photoreceptors under the control of the opsin promoter begin-Ž . ning approximately on day 7 Okamoto et al., 1997 . Retinal degeneration does not occur in other VEGF transgenic lines or other viral infection models, so a precise level and time of VEGF expression may be critical in promoting retinal degeneration.

One of the possible contributing factors in the role of VEGF in EAU and herpesvirus retinopathy and its apparent absence in ECOR is the predominant inflammatory cell entering the retina. EAU is characterized by a primary T cell infiltrate and herpesvirus retinopathy is characterized ( ) Ž by a predominance of T cells and NK cells Tanigawa et . al., 2000 . In contrast, ECOR is characterized by a predominance of monocytes followed by a much smaller T cell infiltrate. Differences in the composition of the cellular infiltrates in these conditions could predispose a different spectrum of soluble factors released, thus accounting for differences in VEGF induction.

VEGF receptors have been identified on monocytes ŽShen et al., 1993; Barleon et al., 1996; Clauss et al., 1996;  . Sawano et al., 2001 and VEGF can stimulate the activa-Ž tion and migration of monocytes Clauss et al., 1990 Clauss et al., , . 1996 ; Barleon et al., 1996; Heil et al., 2000 . However, despite the fact that monocytes are the primary infiltrating cell type, VEGF does not appear to promote monocyte Ž . infiltration in ECOR, since 1 VEGF induction is not Ž . evident until the inflammation has subsided and 2 VEGF induction is not demonstrated in CD-1 mice despite the fact that the inflammatory response to coronavirus infection in CD-1 mice is even greater than that in BALBrc mice.

In models such as EAU and experimental herpesvirus retinopathy, in which VEGF is upregulated early during the course of the disorder, TGFb2 has been postulated to inhibit the angiogenic activity of VEGF, accounting for the Ž absence of neovascularization Vinores et al., 1997a Vinores et al., , . 1998a . Neovascularization also does not occur in ECOR, but without the upregulation of VEGF, an angiogenesis inhibitor may not be necessary to inhibit vascular growth. Thus, there are no differences in the expression of TGFb2 relative to the course of the disease, suggesting that TGFb2 is unlikely to play a role in its pathogenesis.

In summary, BRB breakdown was observed in the acute inflammatory phase of ECOR in both retinal degeneration Ž . susceptible BALBrc and retinal degeneration resistant Ž . CD-1 mice. In comparison to BALBrc mice, the CD-1 mouse BRB breakdown was more extensive and correlated with an enhanced inflammatory response. The findings indicate that BRB breakdown correlates directly with inflammatory cell infiltration and not with retinal degeneration in both sensitive and resistant strains. This study clearly demonstrates that although VEGF plays a basic role in the development of EAU and herpes simplex retinitis, it is not involved in the pathogenic processes in ECOR. Its upregulation, only in mice undergoing retinal Ž degeneration BALBrc mice after the acute inflammatory . response , suggests that VEGF induction may be a secondary change related to retinal degeneration, rather than to inflammation or viral infection. Moreover, the lack of upregulation of VEGF in ECOR may be one of the factors which allows the CD-1 mouse retina to return to a normal appearance after the acute disease.

Categorical Data Analysis

Cross-reaction of a rat-anti-mouse Ž . phagocyte-specific monoclonal antibody anti-Mac-1 with human monocytes and natural killer cells

Migration of human monocytes in response to vascular Ž . endothelial growth factor VEGF is mediated via the VEGF receptor flt-1

Localization of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration

The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis

Vascular permeability factorrvascular endothelial growth factor, microvascular hyperpermeability and angiogenesis

Similarities between the oxygensensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin

Adenosine as an endogenous mediator of hypoxia for induction of vascular endothelial growth factor mRNA in U-937 cells

Vascular endothelial growth factor Ž . VEGF stimulates monocyte migration through endothelial monolayers via increased integrin expression

Retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy

Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia

VEGF increases retinal vascular ICAM-1 expression in vivo

Blood-retinal Ž . barrier BRB breakdown in experimental autoimmune uveoretinitis: comparison with vascular endothelial growth factor, tumor necrosis factor a , and interleukin-1b-mediated breakdown

Test for trend in proportions

Encyclopedia of Statistical Sciences

During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium

Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo

Hypoxia regulatory elements of the human vascular endothelial growth factor gene

Transgenic mice with increased expression of vascular endothelial growth factor in the retina. A new model of intraretinal and subretinal neovascularization

Intravitreal sustained release of VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and primates

Vascular endothelial growth factorrvascular permeability factor expression in a mouse model of retinal neovascularization

Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo

Murine coronavirus induces an acute and long-lasting disease of the retina

Ocular tropisms of murine Ž . coronavirus strain JHM after inoculation by various routes

Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor

Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans

Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid

A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines

Characterization of vascular permeability factorrvascular endothelial growth factor receptors on mononuclear phagocytes

Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis

Natural killer cells prevent direct anterior-to-posterior spread of herpes simplex virus type I in the eye

Assessment of blood-retinal barrier integrity

Immunohistochemical localization of blood-retinal barrier breakdown in human diabetics

Ultrastructural localization of blood-retinal barrier sites in diabetic and galactosemic rats

Localization of blood-retinal barrier breakdown in human pathologic specimens by immunohistochemical staining for albumin

An adenosine agonist and prostaglandin E cause breakdown of the blood-retinal barrier by 1 opening tight junctions between vascular endothelial cells

Electron microscopic immunocytochemical demonstration of bloodretinal barrier breakdown in human diabetics and its association with aldose reductase in retinal vascular endothelium and retinal pigment epithelium

Electron microscopic immunocytochemical evidence for the mechanism of blood-retinal barrier breakdown in galactosemic rats and its association with aldose reductase expression and inhibition

Immunohistochemical localization of blood-retinal barrier breakdown sites associated with post-surgical macular edema

Blood-retinal barrier breakdown in retinitis pigmentosa: light and electron microscopic immunolocalization

Blood-retinal barrier breakdown in eyes with ocular melanoma: a potential role for vascular endothelial growth factor

Upregulation of VEGF and TGFb2 in experimental herpesvirus retinopathy

Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease

Increased Ž . vascular endothelial growth factor VEGF and transforming growth Ž . factor b TGFb in experimental autoimmune uveoretinitis: upregulation of VEGF without neovascularization

Electron microscopic evidence for the mechanism of blood-retinal barrier breakdown in diabetic rabbits: comparison with magnetic resonance imaging

Coronavirus JHM replication within the retina: analysis of cell tropism in mouse retinal cell cultures

Genetic predisposition to coronavirus-induced retinal disease

The role of apoptosis within the retina of coronavirus infected mice

Ocular disease induced in mice by anterior chamber inoculation of herpes simplex virus

This study was supported, in part, by NIH grants EY10017 and EY05951 from the Public Health Service, US Department of Health and Human Services, Bethesda, MD, USA, and by The Lew R. Wasserman Merit Award Ž . SAV and an unrestricted grant from Research to Prevent Blindness. We wish to thank Michele Melia for assistance with statistical analysis.