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Ocular toxoplasmosis in mice: comparison of two routes of infection
Published online by Cambridge University Press: 23 May 2005
Abstract
This paper aims to test the influence of route of infection (intravitreal and instillation) on the course of ocular toxoplasmosis in mice, using the Toxoplasma gondii Me-49 strain. All mice inoculated intravitreally or by instillation presented the same pattern of infection. Using either route, parasites were observed in the retinal vessel with the formation of a glial reaction in the inner plexiforme layer and discontinuity of the pigmented epithelium of the retina 7 days after infection. However, when the intravitreal route was used a more intense inflammatory infiltrate was observed in the retina. The results suggest that inoculation route remarkably influences the inflammatory pattern in ocular toxoplasmosis and that the instillation route should be preferentially used in experimental infections in the murine ocular model of infection by T. gondii, specially with small animals where there is extensive needle damage, which is not observed in the instillation route.
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INTRODUCTION
Toxoplasma gondii is the causative agent of toxoplasmosis. This protozoa is an obligate intracellular parasite that is able to infect and thrive in most nucleated cells of warm-blooded animals causing a variety of clinical syndromes in humans and animals (Frenkel & Jacobs, 1958). Although toxoplasmosis is generally asymptomatic in healthy adults, the infection may cause severe sequelae in neonates and life-threatening lesions in AIDS patients (Ortega-Barria & Boothroyd, 1999). The prevalence of T. gondii -specific antibodies in adults increases with advancing age but differs widely with the geographical location (Lynfield & Guerina, 1997).
Ocular disease in newborns with congenital T. gondii infection was first described in 1923 (Jankû, 1923), although it was not until 1939 that congenital toxoplasmosis with ocular involvement was widely accepted as a human disease (Wolf, Cowen & Paige, 1939). However, ocular toxoplasmosis in adults was not recognized until 1952 (Wilder, 1952). It is known that the eye is an immunologically privileged site (Medawar, 1945, 1948) and retinal pigmented epithelial cells (RPE), at the level of the posterior segment of the eye, play a crucial role in the induction and maintenance of this immune privilege (Sun et al. 2003; Tedesco et al. 2004). Tedesco et al. (2004) showed that RPE cells migrate into the internal retina layer during murine experimental toxoplasmosis. In addition, Tedesco et al. (2004) presented abundant evidence for the crucial role of RPE cell migration in experimental ocular toxoplasmosis.
When congenital ocular toxoplasmosis is present in childhood, the infection may interfere with eye development (Dutton, 1989). Uveitis is initiated in every instance by some form of tissue injury as, for example, the penetration of a single cell by an organism such as Toxoplasma (O'Connor, 1983; Holland, 2004). Several animal models (e.g. mouse, rabbit) have been used to study ocular toxoplasmosis (Garweg, Kuenzli & Boehnke, 1998; Tedesco et al. 2004). Most of the studies in ocular toxoplasmosis use the intraperitoneal (Pavesio et al. 1995) or intravitreal (Garweg, 1998) routes for parasite inoculation. Using T. gondii ME-49 strain the intraperitoneal route promotes a parasite dispersion causing an infection with low parasite loads in the eye (Gormley et al. 1999). The intravitreal route is difficult to perform on small animals, such as mice, frequently resulting in loss of part of the inoculum or dissociation of ocular tissue (Tedesco, 2003).
The aim of this study was verify the influence of route of inoculation (instillation and intravitreal) on the development of T. gondii ocular infection in mice.
MATERIALS AND METHODS
Animals
Thirty-three C57BL/6 female mice at 6–8 weeks of age, weighing about 12–18 g were purchased from the Animal Facility (CECAL) of the Oswaldo Cruz Institute – FIOCRUZ.
Parasites
Tissue-derived cysts from T. gondii, ME-49 strain, were obtained from the brains of previously infected C57BL/6 mice and maintained by serial intraperitoneal (i.p.) passage in C57BL/6 mice.
Cyst isolation
Mice were killed 30 days after infection by CO2 inhalation and their brains were surgically removed, washed with sterile saline and homogenized. The total homogenate was submitted to centrifugation for cyst purification, according to the method of Freyre (1995) and Popiel, Gold & Booth (1996). The pellet was resuspended in 2 ml of sterile saline and 20 μl of suspension were used to evaluate cyst number by counting on a 24×32 mm cover-slip. The cyst walls were disrupted by pepsin/HCl according to the method described by Jacobs, Remington & Melton (1960) and Dubey, Lindsay & Speer (1998), and the parasites counted in an haemocytometer.
Experimental schedule
Animals were divided in 2 groups: Group 1: 15 normal mice were anaesthetized with poximetacaíne cloridrate 0·5% (Alcon Lab. do Brasil LTDA) using 1 drop on each eye. Infection was made according Garweg et al. (1998), with 5×103 bradyzoites in 5 μl of PBS intravitreally, 15 min after anaesthesia; Group 2: 15 normal mice were infected by instillation (i.t.) of 5×103 bradyzoites in 1 drop, according to Gil et al. (2002); Group 3: 3 non-infected control mice. Animals were killed by CO2 inhalation 12, 24, 48 h, 5 and 7 days after infection and the eyes enucleated and sectioned in a sagital plane, dividing the bulb into 2 hemispheres. Both hemispheres were processed for historesin embedding medium (hydroxyethyl-methacrylate, Leica – Heidelberg, Germany).
Serological assays
Antibodies against T. gondii were examined in serum samples by indirect immunofluorescence assay (IIFA) (Camargo, Leser & Leser, 1976) on 12, 24, 48 h, 5 and 7 days after T. gondii infection. Serum was obtained from blood of the experimental animals and diluted in phosphate buffer solution (PBS) from 1[ratio ]4 to 1[ratio ]128 for the IIFA test. The antigen used in the IIFA was a sample of T. gondii isolated from mouse peritoneal exudates. Parasites were killed with 2% formaldehyde and distributed on the slides (40 parasites/field). Secondary antibody conjugated with fluorescein isothiocyanate (FITC), (Sigma, St Louis, USA) (goat anti-mouse IgM or IgG) was diluted 1[ratio ]100 in PBS-Evans's blue. Slides were analysed in a microscope (Axioplan – Zeiss) with epiluminescence illumination by mercury-vapour lamp (HBO-200). Positive serum was obtained from C57BL/6 mice infected with T. gondii, ME-49 strain and negative serum obtained from non-infected C57BL/6 mice.
Experiments were performed in accordance to the rules the Ethic Committee from Institute Oswaldo Cruz – FIOCRUZ.
Historesin preparation
Samples were immersion-fixed for 12 h at 4 °C in 2·5% (v/v) glutaraldehyde diluted in 0·1 M Na-cacodylate-buffer, pH 7·4, containing 3·5% sucrose. The material was dehydrated in a graded ethanol series and embedded for 12 h in 100% ethanol and historesin (glycol methacrylate – GMA-Leica) (1[ratio ]1) with low agitation. Blocks were cut at a thickness of 1 μm in a semi-series (Micron – HM360) and stained with 0·25% toluidine blue in 1% sodium borate. Slides were preserved in Entellan (Merck, Darmstadt, Germany) covered with cover-slips and observed with a microscope (Axioplan – Zeiss).
RESULTS
Control eyes
Examination of normal non-infected mice eye sections, under light microscopy showed normal morphology (Fig. 1A). However, in PBS control (i.v.) animals, eye histology showed a discontinuity in the retinal tissue with focal oedema in the outer segments of photoreceptors, in the outer nuclear layer and outer plexiforme layer. Morphological alterations were also evident in the lens and capsule and apparently related with inoculation site (Fig. 1B). We also observed discontinuity of the pigmented epithelium of the retina and chorioid. These observations were constant and observed in all necropsy periods.
Fig. 1. Light micrograph of eye sections stained with toluidine blue. (A) Control eye from normal non-infected mouse showing the normal architecture of the retina. All layers unaltered: retinal pigmented epithelium (RPE) (arrow); outer segments of photoreceptors (FTR); outer limited membrane (arrowhead); outer nuclear layer (ONL); outer plexiforme layer (OPL); inner nuclear layer (INL); inner plexiforme layer (IPL); ganglion-cell layer (GCL); nerve-fibre layer (NF); chorioid (C); sclera (S). The inner limited membrane is not shown but it had normal architecture. (B) General aspect of the eye submitted by intravitreal inoculation. There is a gap formation between RPE and the outer segment of FTR (*). Alterations in the disposition of lens cells (L) are observed (thick arrow). (C) Instillation inoculation. There is normal disposition of the ONL and INL, outer segment of the FTR and IPL. No alterations in the disposition of the lens cells (L) observed. (D) Intravitreal inoculation with gap formation in the outer segment of FTR (*) and alteration in the disposition of ONL, INL and IPL and lens cells (L) (thick arrow). (E) Parasite observed in the retinal vessel of the instillation inoculated eye (arrow), and glial cells in the IPL (outline arrow), oedema in the ONL and INL (white arrow) and formation of lacunar spaces in outer segments of the FTR (*). (F) Intravitreal inoculation showing the parasite inside the retinal vessel (arrow), presence of inflammatory infiltrate (arrowhead) localized in the vessel and vitreous humour. Glial cells in the IPL were also observed in these mice (outline arrow). (G) The retina presents alterations in the disposition of its layers (*) after 7 days of instillation inoculation. (H) Presence of the parasite inside of the choroid (C) vessel (arrow), edema in the ONL (white arrow) and enlargement of the interstitial space between the segments, forming lacunae (*), 7 days post-intravitreal infection.
Acquired ocular toxoplasmosis in C57BL/6 mice – light microscopy
Instillation infection
Animals submitted to instillation (i.t.) with a drop of PBS containing 5×103 bradyzoites did not present severe alterations in the disposition of ocular structures. The lens was continuous and its capsule preserved. The retina was also continuous and did not present space between its layers (detachment) (Fig. 1C). However, we did observe parasites in the retinal vessels with glial migration into the inner retinal layers and oedema formation in the inner nuclear and ganglionar layers (Fig. 1E). Focal retinochoroiditis was also observed at all infection periods (Fig. 1G).
Intravitreal infection
In mice that received 5×103 tachyzoites i.v. (Group 3) we observed alterations in the disposition of retina layers, appearance of retinal space and discontinuity of pigmented epithelium and choroid, as well as damage of the architecture of lens and capsule (Fig. 1D). Independently, on the infection day, we observed a perivascular, intravascular and vitreo-retinal-interface inflammatory infiltrate, composed mainly of mononuclear and polymorphonuclear cells, associated with an increase of glial cells in the plexiform and ganglionar layer (Fig. 1F). This infiltrate induced oedema characterized by an increase of interstitial spaces, and formation of lacunae in the outer segment layers of the photoreceptors (FTR) and in the outer nuclear and plexiforme layers (Fig. 1H).
Serological evaluation
An indirect immunofluorescence assay was used to confirm infection. Results show that 100% of mice were infected independently of the infective route. Titres varied from 1[ratio ]64 IgM, 12 h after infection; 1[ratio ]128 (24 to 48 h). This titre fell to 1[ratio ]8, 7 days after infection and the IgG titre reached 1[ratio ]4 at this time.
DISCUSSION
Many aspects of toxoplasmic retinochoroiditis have been investigated in man and animals. Naturally occurring ocular toxoplasmosis and the disease induced experimentally by local or systemic injection in animals have been well documented (Lee et al. 1983; Smith & Cunningham, 2002; Holland, 2004). There has been, however, no detailed study of routes of infection in an attempt to find the best route for T. gondii infection in experiments using small animals.
In this work, the results showed that both intravitreally and instillation infection routes were able to produce progressive ocular toxoplasmosis in mice. We did not detect differences in parasitism among individuals inoculated by either of the two routes. In both we observed parasites in the vessels, with glial reaction in the inner plexiforme layer and discontinuity of the pigmented retinal epithelium after 7 days of infection. These alterations were also observed by Tedesco et al. (2003) 60 days post-infection using the intraperitoneal route. In spite of that, intravitreal inoculation of PBS without parasites also caused detectable alterations of the retina. The intravitreal route is generally used in larger animals (Yoshizumi, 1976; Garweg et al. 1998) and was used in this study to establish a direct contact between the parasite and the mouse retina.
Our results showed that intravitreal inoculation caused an ocular involvement, observed in the histopathological analysis, originated by the invasive procedure of inoculation. This may be because the mouse eye is small and the mechanical trauma in the inoculation site causes detachment of the retina by increasing the intraocular pressure. These mechanical effects are in addition to the presence of parasites (retinochoroiditis), which are normally associated with vitrial inflammatory infiltrate of different grades. These results confirm the findings of Garweg et al. (1998), who used the same route of infection (intraocular) with tachyzoites of BK strain in rabbits.
Our results suggest that the use of the intravitreal inoculation route leads to a localized lesion in the lens and in disposition of the retinal layers. This results in detachment of the retina that can be attributed to the inoculation site and increased intraocular pressure caused by the volume of the inoculum. These changes are not observed by using inoculation by instillation. The retinochoroiditis, vasculitis and glial reaction observed in the inner plexiforme layer are clearly attributed to parasite infection, independent of inoculation route.
In conclusion, we suggest that the instillation route should be preferentially used in experimental toxoplasmosis in the murine ocular model, to obtain an ocular infection in a short time. This route is preferably better than the intravitreal route, particularly because the increased intraocular liquid volume and inflammatory infiltrate caused by injection are not observed using the instillation route.
The authors thank Dr Fabio Santori for suggestions and revision of the English. This work was supported by a grant from the Instituto Oswaldo Cruz – Fundação Oswaldo Cruz.
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Fig. 1. Light micrograph of eye sections stained with toluidine blue. (A) Control eye from normal non-infected mouse showing the normal architecture of the retina. All layers unaltered: retinal pigmented epithelium (RPE) (arrow); outer segments of photoreceptors (FTR); outer limited membrane (arrowhead); outer nuclear layer (ONL); outer plexiforme layer (OPL); inner nuclear layer (INL); inner plexiforme layer (IPL); ganglion-cell layer (GCL); nerve-fibre layer (NF); chorioid (C); sclera (S). The inner limited membrane is not shown but it had normal architecture. (B) General aspect of the eye submitted by intravitreal inoculation. There is a gap formation between RPE and the outer segment of FTR (*). Alterations in the disposition of lens cells (L) are observed (thick arrow). (C) Instillation inoculation. There is normal disposition of the ONL and INL, outer segment of the FTR and IPL. No alterations in the disposition of the lens cells (L) observed. (D) Intravitreal inoculation with gap formation in the outer segment of FTR (*) and alteration in the disposition of ONL, INL and IPL and lens cells (L) (thick arrow). (E) Parasite observed in the retinal vessel of the instillation inoculated eye (arrow), and glial cells in the IPL (outline arrow), oedema in the ONL and INL (white arrow) and formation of lacunar spaces in outer segments of the FTR (*). (F) Intravitreal inoculation showing the parasite inside the retinal vessel (arrow), presence of inflammatory infiltrate (arrowhead) localized in the vessel and vitreous humour. Glial cells in the IPL were also observed in these mice (outline arrow). (G) The retina presents alterations in the disposition of its layers (*) after 7 days of instillation inoculation. (H) Presence of the parasite inside of the choroid (C) vessel (arrow), edema in the ONL (white arrow) and enlargement of the interstitial space between the segments, forming lacunae (*), 7 days post-intravitreal infection.
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