|Molecular Vision 2004;
Received 25 April 2004 | Accepted 23 December 2004 | Published 30 December 2004
Albumin is not present in the murine interphotoreceptor matrix, or in that of transgenic mice lacking IRBP
1Department of Ophthalmology, University of Virginia, Charlottesville, VA; Departments of 2Ophthalmology, 3Pathology, and 4Biochemistry, Ross Eye Institute, State University of New York, Buffalo, NY; 5Medical Research Service, Veterans Affairs Medical Center, Buffalo, NY
Correspondence to: Federico Gonzalez-Fernandez, MD, PhD, Medical Research
Service, Veterans Affairs Hospital, Buffalo, NY, 14215; Phone: (716)
862-6519; FAX: (716) 862-6526; email: firstname.lastname@example.org
Dr. Liao is now at the Department of Ophthalmology, Anhui Medical University, Hefei, Anhui, Republic of China.
Purpose: The interphotoreceptor matrix mediates interactions between the retinal pigment epithelium, photoreceptors, and Müller cells. Each of these cells contributes to specific proteoglycans, proteins, and growth factors in the interphotoreceptor matrix. Some components, such as interphotoreceptor retinoid binding protein (IRBP), are virtually unique to the interphotoreceptor matrix. It has been proposed that serum albumin, thought to be present in the interphotoreceptor matrix, could act as a surrogate retinoid binding protein within the subretinal space of transgenic mice lacking interphotoreceptor retinoid binding protein. To address this question, we sought to determine whether albumin is present in the interphotoreceptor matrix of IRBP+/+ mice or IRBP-/- mice.
Methods: We examined the distribution of albumin in IRBP-/- mice and IRBP+/+ mice using immunofluorescence and immunoperoxidase histochemistry.
Results: The distribution of albumin within the corneal stroma, sclera, and capillaries is consistent with previous work. Serum albumin could not be detected in the interphotoreceptor matrix. The distribution of albumin in IRBP-/- mice was similar to that of their wildtype counterparts.
Conclusions: Serum albumin is not a component of the interphotoreceptor matrix of IRBP+/+ mice or IRBP-/- mice.
Among the various ocular structures found in the animal kingdom, the eyes of vertebrates employ an innovative design consisting of a two layered cup. Posteriorly, the two layers are separated by the subretinal space [1-3]. Unlike the faceted ommatidial structure of the insect eye, or the inside out arrangement of the cephalopod retina, vertebrate photoreceptors form a single sheet in apposition to a continuous layer of cuboidal epithelium, the retinal pigmented epithelium (RPE). Separating the RPE from the neural retina is an extracellular material termed the interphotoreceptor matrix (IPM). Sclerad, the IPM is bordered by the RPE whose zonulae occludens help to form the choroidal blood-retinal barrier. Vitread, the IPM is bordered by the photoreceptors and Müller cells. These cells are separated by zonulae adherens, which prevent molecules with a Stokes' radius >30 Å from diffusing out of the subretinal space . Thus, the photoreceptors, RPE, and Müller cells have access to each other through the IPM whose border they share. This anatomic arrangement allows for interactions between each of these three cell types.
An example of such a collaboration is the role of photoreceptors, RPE, and Müller cells in the exchange of visual-cycle retinoids. The IPM is thought to facilitate this exchange through the presence of retinoid binding proteins. In this regard, most of the attention has focused on a 135 kDa (human) glycolipoprotein termed interphotoreceptor retinoid binding protein (IRBP) [5-8]. IRBP, which is secreted by the rods and cones, accumulates in the IPM because its large size prevents it from exiting the subretinal space through the zonulae adherens. The finding that retinoid trafficking in vitro  and in vivo  is uninterrupted in the absence of IRBP, and in fact appears to be accelerated [9,11,12], suggests that the main function of IRBP is not simply to solubilize retinoids. Its more important role may be to regulate the cellular release of retinoids [13-16], protect retinoids from degradation [15-18], protect cells from retinoid toxicity , and to buffer free retinoids . Finally, the proposal that IRBP has a role in the exchange of 11-cis and all-trans retinol between the cones and Müller cells needs to be explored .
The IPM is a complex structure, and may contain additional proteins capable of binding retinoids. These candidates have not been as thoroughly studied as IRBP in regards to their localization or function in the retina. It should be kept in mind that typical IPM extracts may contain contaminating serum proteins and proteins that have leaked from damaged cells. Furthermore, it is plausible that some proteins may be secreted into the IPM, but do not accumulate in the subretinal space because their small size allows them to cross the zonulae adherens. This may be the case for serum retinol binding protein, which is thought to be secreted by the RPE .
In the present study, we wanted to determine the localization of serum albumin in the mouse eye with particular attention to the IPM. There are several reasons that we thought such a study would be helpful towards understanding the structure and function of the IPM. First, there is some controversy as to whether the IPM normally contains serum albumin. Although serum albumin is often detected as a major band by SDS-PAGE of soluble extracts of the IPM [22-29], the source(s) of this albumin has not been thoroughly investigated. It is possible that this albumin reflects serum proteins contaminating the matrix extract during the tissue dissection rather than the actual normal composition of the IPM. Nevertheless, the mouse retina is capable of producing serum albumin since albumin mRNA can be detected in the mouse neural retina by real time RT-PCR . Whether albumin is normally present in the IPM is not clear as it has been detected in the subretinal space in some  but not all studies [30-34]. Our second motivation for performing this study is that it has been proposed that albumin may transport visual-cycle retinoids in the IPM . This notion has been offered as an explanation for the nearly normal visual cycle in transgenic mice lacking IRBP . According to this idea, albumin acts as a surrogate-retinoid carrier in the absence of IRBP. However, no study has sought to determine if albumin is present in the IPM of IRBP-/- mice. Here, we used immunofluorescence and immunoperoxidase approaches to define the distribution of albumin in the eyes of normal mice, and in those of transgenic mice lacking IRBP.
Our study was approved by the Animal Care Committee of the University of Virginia, where the work was begun, and by the Medical Research Service of the Veterans Affairs Health System and State University of New York where the work was continued. All animals used in these experiments were maintained according to The ARVO Resolution on the Use of Animals in Research. All animals were housed under a 12 h light/12 h dark room-light cycle. Wild type C57B1 mice were obtained from Harlan (Indianapolis, IN). Transgenic mice lacking IRBP (IRBP-/-), which are in a C57B1 background, have been previously described .
As ocular tissues often exhibit autofluorescence, we took two precautions to distinguish specific from nonspecific background signal. First, the distribution of albumin was studied not only by direct immunofluorescence, but also by avidin-biotin immunoperoxidase histochemistry . Complete histological cross-sections of the globe were prepared from animals 11 days, 25 days, and 2.5 month of age. For immunofluorescence studies, the sections were incubated with an FITC conjugated rabbit IgG directed against mouse albumin. For the immunoperoxidase studies, the primary antibody was an unlabeled rabbit anti-mouse albumin IgG, which was localized using a biotinylated goat anti-rabbit IgG. As a second control for nonspecific background signal, we performed parallel incubations in which the primary antibody had been preadsorbed with purified mouse albumin.
Localization of albumin by direct immunofluorescence
Eyes were enucleated immediately after death. To facilitate entry of fixative into the globes, an incision was made through the central cornea avoiding the angle structures. The eyes were then submerged in freshly prepared 4% paraformaldehyde with 5% sucrose in 100 mM sodium phosphate at pH 7.4 and incubated overnight at 4 °C with gentle agitation. The eyes were gradually infiltrated into 50% optimal cutting temperature compound (Tissue-Tek OCT, Electron Microscopy Sciences, Fort Washington, PA) and 10% sucrose in 100 mM sodium phosphate at pH 7.4, and frozen under 100% OCT in 2-methylbutane chilled with liquid nitrogen. Cryosections were cut in a sagital orientation at 10 μm, placed on charged slides (X-tra, Surgipath, Richmond, IL), and held at -80 °C until use.
The sections were incubated with an FITC conjugated rabbit IgG directed against mouse albumin (Cederlane, Hornby, Ontario, Canada). To distinguish background fluorescence and autofluorescence from immunospecfic fluorescence, preadsorbed controls were included in every experiment . These controls consisted of pre-incubating the antibody with purified mouse albumin (Sigma Aldrich, St. Louis, MO). Background and autofluorescence are not changed in the preadsorbed control. In contrast, fluorescence due to a specific interaction between tissue albumin and the antimouse albumin FITC conjugated IgG is abolished in the preadsorbed control section. Sections were finally mounted in Fluoromount-G mounting medium (Southern Biotechnology, Birmingham, AL). Fluorescence microscopy was performed on an Axioplan 2 microscope (Zeiss, Thornwood, NY) equipped with a high sensitivity Orca-1 CCD camera system (Hamamatsu, Bridgewater, NJ), and imaging software (MetaMorph, Universal Imaging, West Chester, PA). The laser scanning confocal microscope consisted of a Nikon PCM2000 coupled to a TE-200 epifluorescence microscope which used a Orca-1 Hamamatsu Camera.
Localization of albumin by immunoperoxidase histochemistry
Excised globes were fixed as described above in 4% paraformaldehyde in 100 mM sodium phosphate at pH 7.4, and processed for paraffin embedding. Sections (5 μm) were cut, deparaffinized, and rehydrated according to standard protocols. The sections were then incubated with primary antibody (rabbit anti-mouse serum albumin; Bethyl Laboratories, Montgomery, TX) for 2 h at room temperature. The sections were washed 3 times in PBS and incubated with secondary antibody for 1 h (goat anti-rabbit biotinylated IgG; Vector Labs, Burlingame, CA). The sections were washed again, incubated with avidin-biotin complex, and developed wth DAB. In adsorbed controls, the sections were treated in an identical manner except that the primary antibody was pre-incubated with purified mouse albumin.
Figure 1 shows the distribution of albumin in the normal IRBP+/+ retina at p25. For this figure, and all of the other immunofluorescence studies below, we use a purified FITC conjugated rabbit IgG directed against mouse albumin. Immunospecific fluorescence was consistently associated with the sclera and capillaries, and was abolished by omitting the antibody or by preadsorption of the antibody with purified mouse albumin (Figure 1A,B). Immunospecific fluorescence was detected in the choroid although fluorescence in this region was partly masked by the melanin pigment. The rod outer segments, erythrocytes, and the extraocular muscles often showed endogenous background fluorescence. Some autofluorescence is not uncommon for these structures . However, comparisons with the preadsorbed controls clearly distinguishes the genuine immunospecific fluorescence from this nonspecific autofluorescence. For example, in Figure 1B the autofluorescence associated with muscle (top of panels above asterisk), and the outer segment layer was not reduced by antibody preadsorption with mouse albumin. In contrast, antibody preadsorption abolishes fluorescence in the sclera, retinal vessels, and choroid (Figure 1B). Figure 1C shows the retina at higher magnification with the outer segments oriented in cross-section. In this orientation, it is evident that the autofluorescence is restricted to the outer segments, with little fluorescence associated with the space between the outer segments. This pattern and degree of fluorescence is unchanged in the preadsorbed control (Figure 1D). We found no evidence for immunospecific fluorescence associated with the IPM by fluorescence microscopy, or by laser scanning confocal microscopy.
Albumin was also consistently detected in the corneal stroma. This result is illustrated in Figure 2 for a normal C57B1 mouse at 2.5 month of age. The albumin was restricted to the stroma and did not appear to be present in the corneal epithelium or endothelium. The stromal fluorescence was abolished by omitting the antibody, or by preadsorbing it with purified mouse albumin (Figure 2C-F).
We next wanted to know if the distribution of albumin is different in IRBP-/- mice. Specifically, is there evidence for albumin entering the subretinal space perhaps secondary to the pathological changes in this transgenic mouse? Of particular interest were p25 animals as biochemical studies of retinoid transport in the IRBP-/- mouse have been performed using animals at about this age . As in previous figures, the sections in Figure 3 were treated with FITC conjugated antimouse albumin IgG (Figure 3A,B), no antibody (Figure 3C), or antibody preadsorbed with mouse albumin (Figure 3D). Immunospecific fluorescence was easily shown in retinal vessels, and the other locations where albumin was found in normal mice. In contrast, albumin could not be detected in the IPM of the IRBP-/- mice.
In Figure 4 and Figure 5 we examined the distribution of albumin in the IRBP-/- and IRBP+/+ mice using peroxidase based immunohistochemistry. The results were consistent with the fluorescence based method. Figure 4 shows the immunohistochemical localization of albumin in anterior structures of the IRBP-/- mouse eye. Albumin was easily identified in the corneal-limbal stroma, capillaries of ciliary body, and retinal-blood vessels where it has been described previously. In constrast, no immunospecific staining could be associated with the IPM. Figure 5 confirms this conclusion in a side-by-side comparison of the distribution of albumin in the posterior retina. Specific staining was easily appreciated in the scleral and retinal vessels, but not in the region corresponding to the IPM in either the IRBP+/+ or the IRBP-/- retina.
Although serum albumin is known to be present in a variety of ocular structures, there are conflicting reports regarding its presence in the IPM. The issue is of further interest as it has been suggested that albumin within the IPM could act as a surrogate for IRBP absent in IRBP-/- mice . However, we found that albumin does not accumulate in the subretinal space of either normal or IRBP-/- mice.
We detected albumin in a variety of structures where albumin has been previously identified. As noted by others, albumin is associated with the iris stroma [32,39-41] and ciliary body stroma [31,39]. Furthermore, our finding that albumin is present in the corneal stroma, but not the corneal epithelium, is consistent with the literature [39,42,43]. In our study, sufficient resolution did not permit evaluating with certainty whether albumin is present in the corneal endothelium. Albumin was also clearly demonstrated in the sclera consistent with previous studies . The function of albumin within these ocular structures is not completely understood. Extravesated albumin from the iris is believed to flow to the angle where is contributes to the trabecular outflow resistance [44,45].
Although albumin is commonly noted on SDS gels of crude IPM extracts [22-29], there are indications in the literature that the source of this albumin is not the IPM itself. Very important in this regard are the careful microcanulation experiments of the monkey subretinal space . In that study a 0.3 mm tube was inserted transclerally into the subretinal space of monkey globes. This allowed, a very gentle detachment and irrigation of the subretinal space that did not appear to damage the detached neural retina or RPE. Using this gentle subretinal wash procedure, the profile of proteins within the extract IPM was very restricted, and different than that of the serum. Furthermore, IRBP was found to be the most abundant soluble protein in the subretinal space. In fact, a definite band in the Mr region for albumin cannot be appreciated from the published photographs . A second older study relevant to the source of albumin in the subretinal space is the careful profiling of proteins in crude IPM extracts from various vertebrates . That study noted a difference in the relative amounts of albumin and IRBP in washes of the detached neural retina and the RPE/eyecup. Typically, albumin was present mainly from the RPE/eyecup wash. In contrast, IRBP was found in both the retina and RPE/eyecup washes. This may represent regional differences in the distribution in the IPM. The alternative interpretation is that the data represent leakage of serum proteins from the choroid. In fact, the choroid is the source of serum proteins in serous retinal detachments .
Our finding that albumin is not present in the IPM of the mouse retina is consistent with some previous studies. Although there is no study focusing on the normal distribution of albumin in the mouse retina, some data on the normal retina is included in a study of the blood-retinal barrier in viral induced murine uveitis. In that study, albumin was not normally associated with the IPM . In rats, albumin is not normally present in the IPM by either immunohistochemical or immunoelectron microscopy [30,33]. Furthermore, intravenous FITC-albumin does not gain access to the outer retina of monkey or rabbits . Albumin is not present in the monkey retina by Evans blue fluorescence microscopy .
Although albumin does not appear to be present in the mammalian subretinal space, our data do not necessarily contradict a recent study that detected albumin in the human IPM , or that albumin mRNA can be detected in the mouse retina . First, the detection of albumin in the human retina by immunofluorescence may represent a species difference. There is precedent that the mouse IPM does not contain other components present in the IPM of most vertebrates . Second, it should be kept in mind that establishing the normal location of albumin in the human retina is not trivial, since postmortem changes in the distribution of albumin occur rapidly . Third, albumin is known to enter the subretinal compartment under some pathological states (see below). Since no clinical information is available on the human eyes used in the study where albumin was detected in the IPM , it is difficult to know whether the presence of albumin may represent preexisting pathology. Finally, serum albumin mRNA can be detected in the mouse retina , and other extrahepatic sites including the brain [49-52]. The identity of the specific cell types responsible for albumin mRNA expression in the retina are not known. Although albumin can be detected in crude extracts of the mouse IPM , and that of other species, it is not clear whether this albumin originates in the IPM (see discussion above). Although albumin mRNA may be expressed in the mouse retina, the results presented here and those reviewed above suggest that albumin does not enter the subretinal space.
The presumed presence of serum albumin in the IPM has led to the suggestion that albumin acts as a surrogate binding retinoids in the IPM of IRBP-/- mice . Although we found that albumin is not present in the normal mouse IPM, we considered that albumin may enter the subretinal space of the IRBP-/- mouse since it is known that albumin may be present in the IPM of some human pathological states, and rodent models of retina disease. For example, in human serous retinal detachments albumin accumulates within the expanded subretinal space . Furthermore, a study localizing the blood-retinal barrier breakdown in a survey of human pathological specimens showed that albumin was sometimes found between the photoreceptor outer segments, and within the RPE in some disease states, but not in normal controls eyes . The potential for sequestration of albumin in the subretinal compartment is due in part to the exclusion limit of the zonulae adherens . The pore radius of these junctions is sufficiently small to prevent the diffusion of albumin into the retina from the IPM. Although too large to diffuse through the zonulae adherens, it can nevertheless leave the subretinal compartment. FITC-albumin in experimental subretinal detachments readily enters the vitreous . Finally, in galactosemic rats, albumin is often seen in the subretinal space and in the intercellular space between photoreceptors . Albumin has also been observed to enter the subretinal space during experimental autoimmune uveoretinitis . We found that albumin is absent from the subretinal compartment of IRBP-/- mice. Furthermore, the distribution of albumin in the various nonretinal ocular structures was overall similar to that of the wild type control mice. In conclusion, although pathological states can disrupt the blood retinal barrier, it does not appear that the absence of IRBP compromises the barrier sufficiently to allow albumin to enter the IPM.
The authors would like to thank Dr. Gregory Liou for providing the transgenic mice used in this study. Supported by National Institutes of Health Grant EY09412 (FG-F).
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