August 2004
Volume 45, Issue 8
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Retinal Cell Biology  |   August 2004
Cultured Embryonic Retina Systems as a Model for the Study of Underlying Mechanisms of Toxoplasma gondii Infection
Author Affiliations
  • Antonio M. M. Moraes
    From the Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
  • Cristiano N. Pessôa
    From the Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
  • Rossiane C. Vommaro
    From the Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
  • Wanderley De Souza
    From the Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
  • Fernando G. de Mello
    From the Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
  • Jan Nora Hokoç
    From the Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil.
Investigative Ophthalmology & Visual Science August 2004, Vol.45, 2813-2821. doi:10.1167/iovs.04-0177
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      Antonio M. M. Moraes, Cristiano N. Pessôa, Rossiane C. Vommaro, Wanderley De Souza, Fernando G. de Mello, Jan Nora Hokoç; Cultured Embryonic Retina Systems as a Model for the Study of Underlying Mechanisms of Toxoplasma gondii Infection. Invest. Ophthalmol. Vis. Sci. 2004;45(8):2813-2821. doi: 10.1167/iovs.04-0177.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Toxoplasma gondii, the most common cause of retinochoroiditis in humans, is an obligate intracellular protozoan parasite that depends on te host cell’s microenvironment to proliferate. Because congenital infection is associated with a higher risk of ocular involvement than a postnatally acquired infection, this study was conducted to investigate the ability of Toxoplasma gondii to infect retinal tissue during development, when cellular environmental changes normally occur.

methods. Retinas from 5- to 9-day-old chick embryos were used. Stationary cultures were prepared in 24-well cell culture dishes and maintained at 37°C in DMEM plus 5% fetal bovine serum for 2 to 6 days. Then the wells were infected with 4 × 105 tachyzoites. Retina explants and aggregate cell cultures were maintained in DMEM under rotation at 37°C. T. gondii proliferation was measured using [3H]-thymidine incorporation after 72 hours. Ornithine and arginine decarboxylase (ODC and ADC) activities were determined by measuring CO2 production from [1-14C]-ornithine and [1-14C]-arginine, respectively.

results. The proliferation of tachyzoites was high in dense, stationary cultures expressing elevated ODC and ADC activity. The addition of ODC or ADC inhibitors reduced T. gondii proliferation by approximately 20% to 40%. As for cultured retina cells, retina explants also allowed T. gondii proliferation whenever ODC activity was high.

conclusions. The data suggest a direct correlation between retinal polyamine biosynthesis and the proliferation of T. gondii, in agreement with the observation that individuals infected congenitally have a greater risk of development of toxoplasmic retinochoroiditis.

The protozoan Toxoplasma gondii is responsible for a worldwide zoonosis. This obligate intracellular parasite infects herbivorous, omnivorous, and carnivorous animals 1 and causes one of the most prevalent chronic infections, affecting 30% to 60% of the world’s human population. 2 Usually asymptomatic, 3 it can become a serious disease when infection is transplacental 4 5 or in immunocompromised individuals. 6 7 8  
T. gondii is the major protozoan that causes intraocular inflammation, being the etiological agent for posterior uveitis in up to 85% of cases. 9 10 Congenital infection is associated with a higher risk of ocular involvement than a postnatally acquired one. 11 12 Retinochoroiditis is the most common finding (58%–82%) in congenital toxoplasmosis 13 14 but is less frequently found in acquired infections. 9 10 13 15 16 17 18 19 20 21 22 23 The most severe complications are observed in the immunocompetent patient and may lead to permanent loss of visual acuity. 24  
It has been difficult to study retinochoroiditis in laboratory animals because several variables may interfere with parasite proliferation and viability. Among these variables the one most frequently found is through protozoan inoculation (intraocular, intraperitoneal, conjunctival or parenteral administration), which could cause an inconstant replication of the parasite in ocular tissue or the premature death of experimental subjects. 25 26 27 28 29 30 31 Some investigators have successfully used primate models, but the high costs inherent in this model makes it prohibitive. 32 33 Hu et al. 34 using intracameral inoculation in the murine model obtained a level of ocular inflammation that seemed to depend on the number of tachyzoites inoculated. Alternative experimental models of congenital toxoplasmosis have also been described in the literature, 35 36 37 38 39 including those intended to investigate aspects of the initial events leading to the passage of T. gondii from maternal to embryonic cell populations. 40  
Only a few studies of the infection of central nervous system (CNS) cells in vitro have been performed. 41 42 43 44 45 These studies demonstrated that the T. gondii tachyzoites infect brain neuronal and glial cells in primary cultures, 41 and that both types of cells allow encystation in the absence of T-cell–derived cytokines. 42 In addition, a greater efficiency in infecting glial cells than infecting neurons was observed. 43 However, there is no report concerning the parasite’s behavior under drastic changes that normally occur in the CNS microenvironment during development. 
T. gondii requires molecules from its host to maintain proliferation and differentiation. A class of molecules named polyamines, plays essential roles in different parasitic protozoa, mainly in the biosynthesis of macromolecules and the control of cell proliferation. 46 47 Because the rate-limiting enzyme for the biosynthesis of polyamines, ornithine decarboxylase (ODC), is absent in this protozoan, 48 polyamines or their direct intermediates should be obtained from the host tissue. 
In the chick retina, ODC activity, which catalyzes the formation of putrescine from ornithine, and putrescine concentration display considerable changes during development, being higher in retinas from younger embryos than in the differentiated tissue. 49  
In this study, we used in vitro models of retinal tissue cultures to evaluate the susceptibility of the tissue to T. gondii infection and proliferation during development. 
Material and Methods
Experimental Models
Fertilized White Leghorn eggs were obtained from a local hatchery. The embryos were staged according to Hamburger and Hamilton 50 and killed by decapitation. Posthatch animals were first anesthetized with ether and then decapitated. 
Swiss mice were housed in the animal facility in the Laboratório de Ultraestrutura Celular Hertha Meyer and were euthanatized by CO2 inhalation. 
Use of animals conformed to the guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the commission of animal care of the Universidade Federal do Rio de Janeiro (UFRJ). 
Parasites
Mice were intraperitoneally inoculated with tachyzoites of T. gondii (RH strain). After 2 to 3 days, a peritoneal wash was performed. The suspension resulting from these washes was centrifuged at 100g for 5 minutes at 4°C and the supernatant, containing viable tachyzoites, was collected and further centrifuged at 1000g for 10 minutes. The parasites were resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco, Grand Island, NY) without fetal bovine serum (FBS) and counted in a Neubauer chamber. 
Tissue Preparation
The eyeballs from White Leghorn embryos and posthatch animals were enucleated and hemisected with a razor blade. The posterior eye cup containing the retina was immersed for 1 hour in fixative solution (4% formaldehyde in 0.16 M phosphate buffer [pH 7.2]). After several rinses in phosphate buffer (PB), the tissue was cryoprotected in 10%, 20%, and 30% sucrose in PB. Then, it was embedded in tissue matrix (Lipshaw; GMI, Albertville, MN), frozen, and sectioned in radial sections (perpendicular to the vitreous surface). Sections (15 μm) were collected in gelatinized slides and stored at −20°C. 
Cell and Tissue Cultures
Retinas from 5-, 8-, and 9-day-old chick embryos (embryonic day [E]5, E8, and E9) were used. For dense, stationary, and aggregate cultures, retinas were digested with trypsin (0.05% in calcium-magnesium free [CMF] solution) and after a brief centrifugation the retinas were dissociated in DMEM-5% FBS by mechanical aspiration with a large-bore pipette. For stationary cultures, the dissociated cells were plated in 24-well culture dishes and maintained in an incubator at 37°C in an atmosphere of 95% air and 5% CO2. When dense, stationary cultures were prepared for immunostaining, the cells were plated on poly-l-lysine–coated coverslips, which were maintained in 24-well plates. The number of cells seeded was 13.5 × 106, 6.3 × 106, and 6.6 × 106 per well in 0.5 mL of DMEM-5% FBS for E5, E8, and E9 retinas, respectively. 
For explant cultures, retinas were cut into segments of approximately 1 to 2 mm2 and maintained in DMEM-5% FBS with rotation (80 rpm in a waterbath at 37°C). Usually, three retinas were used from E5, whereas only one each was used from E8 and E9, in 7 mL DMEM-5% FBS. The cultures were maintained in an atmosphere of 95% air and 5% CO2. For aggregate cell cultures, six E9 retinas were dissociated as described and diluted in 20 mL DMEM-5% FBS. The cell suspension was maintained in air-CO2, at 37°C with rotation (80 rpm). The culture medium was changed every 2, 3, or 4 days in stationary, explant, and aggregate cultures, respectively. 
The dense cultures were maintained in an incubator until confluence (i.e., 2, 5, and 6 days for E5, E9, and E8, respectively) resulting in E5C2, E9C5, and E8C6 cultures, respectively. The explants followed the same scheme for dense, stationary cultures. The aggregate cultures were maintained for 8 days until further use. 
Interaction between T. gondii and Retinal Cells
Cell–parasite interactions were performed in DMEM without FBS (in a 5% CO2-95% air atmosphere at 37°C), for 1 (dense, stationary, and aggregate cultures) or 2 hours (explant cultures), using a 16:1 retinal cell-tachyzoite ratio in stationary and explant cultures or 1560:1 in aggregate cultures. 
Estimation of the Proliferation of T. gondii in Dense, Stationary, and Explant Cultures
After 2, 5, and 6 days in the incubator, cultures prepared from E5, E9, and E8 embryos, respectively, were simultaneously infected. The medium containing FBS was discarded and cultures rinsed in DMEM containing enough parasites to reach the cell (tachyzoite ratios described earlier). After interaction, the cultures were washed twice with DMEM without FBS and then incubated in DMEM-5% FBS containing 0.2 μCi/mL 3H-thymidine. 
At 24, 48, and 72 hours after the initial interactions, sister infected and noninfected dense, stationary cultures supernatants were collected, and each well was washed with PBS. The collected washes were pooled and the final volume filtered under negative pressure through filters (GF/A Whatman; Clifton, NJ) that were further washed to remove nonincorporated thymidine. Infected and noninfected explant cultures were centrifuged, the supernatant collected, and the pooled tissue washed twice. The pooled supernatants were also filtered. The radioactivity was measured by liquid scintillation. Parasite growth was easily evaluated by subtracting 3H-thymidine incorporation in noninfected from that in infected culture supernatants. 
For aggregate cultures, tachyzoites were added to the cell suspension just after mechanical dissociation, which then were maintained with rotation as described earlier. After at least 8 days in the incubator the medium was discarded and the cell aggregates washed and submitted to histologic procedures described for light microscopy of retinal tissue. They were stained with Giemsa, dehydrated in acetone-xylol, and mounted (Entellan; Merck, Darmstadt, Germany). For electron microscopy analysis, the aggregates were washed and rinsed in fixative solution containing 2% paraformaldehyde plus 2.5% glutaraldehyde in PBS. 
Drug Treatment
Polyamine synthesis inhibitors used in this study were added to the incubation medium 24 to 48 hours before host cell–parasite interaction and just after the parasites were removed, after appropriate interaction. 
Assay of ODC and ADC Activities
ODC and arginine decarboxylase (ADC) activities were measured in noninfected dense, stationary cultures (E5C2, E8C6, and E9C5). ADC activity was also evaluated in intact retinas from 7-, 9-, and 14-day-old chick embryos. The activities of these enzymes were estimated by the production of 14CO2 after appropriate incubation of homogenized biological material in the presence of the respective substrates as previously described. 49 51 The protein level was determined by Bradford assay. 52  
ODC Immunostaining
The polyclonal antiserum against ornithine decarboxylase (ODC; Accurate, Westbury, NY) was used. Immunochemistry was performed on retinal cultures and on radial cryostat sections (i.e., sections cut perpendicular to retinal layers). The controls were obtained by omission of the primary antibody, which resulted in the complete absence of labeling. 
Sections.
The retinas were then preincubated in 3% normal goat serum in phosphate-buffered saline (PBS; pH 7.4) and 0.25% Triton X-100 for 30 minutes and then incubated in primary antibody solution overnight at room temperature. ODC antiserum was diluted 1:500 in PBS with 0.25% Triton X-100. The immunoreactivity was visualized using the avidin biotin peroxidase complex method (ABC; Vector Laboratories, Burlingame, CA), using diaminobenzidine (DAB) as a chromogen. Briefly, the tissue was incubated in biotinylated goat anti-rabbit IgG diluted 1:50 in PBS with 0.25% Triton X-100 for 2 hours, rinsed, and incubated in the avidin-peroxidase complex diluted 1:100 in PBS for 30 minutes. The sections were incubated in DAB (0.1 mg/mL) and H2O2 (0.05%) in Tris buffer (0.1 M, pH 7.6) for 10 minutes (the reaction time was monitored under a microscope). Rinsing several times in PBS interrupted the reaction, and the retinas were mounted on glass slides in 40% glycerol in phosphate buffer and coverslipped. 
Cultures.
Dense, stationary cultures were washed three times in PBS (10 minutes each) and then fixed with 4% paraformaldehyde in PBS for 10 minutes. Subsequent to other PBS washes, the coverslips were incubated with a blocking solution composed of 3% bovine serum albumin (BSA; Sigma-Aldrich, MO) and 0.01% Tween-20 (Bio-Rad, Hercules, CA) for 30 minutes, followed by overnight incubation with ODC polyclonal antiserum (Accurate) diluted 1:500. The cultures were rinsed twice in PBS (5 minutes each) and subsequently incubated with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich) for another 2 hours. After two rinses (5 minutes each) in PBS, the immunocytochemistry was revealed by the blue SG chromogen (Vector Laboratories) for 15 minutes, followed by further PBS rinses. All the incubations with the antibodies were performed under continuous gentle agitation, and the antisera were diluted in the blocking solution. 
Statistical Analysis.
All data are expressed as the mean ± SEM. The overall statistical significance was first obtained by one-way analysis of variance (ANOVA). The statistical significance of all pairs of multiple groups of data was assessed by the Bonferroni comparison test. P < 0.05 was considered significant. 
Results
Evaluation of Parasite Growth
Three confluent dense, stationary cultures were simultaneously infected: two of them at the developmental stage equivalent to E14 when low polyamine levels are observed (E8C6 and E9C5 ≅ E14) and one at a retinal equivalent stage E7 (E5C2) with high polyamine content. 49 53 Unexpectedly, tachyzoite production was high in cultures from older embryos (E14 equivalent) and less prominent in cultures from young embryos, as assessed by [3H]-thymidine incorporation (Fig. 1) . Thus, parasite growth in E9C5 cultures was five times higher than that in E5C2, 72 hours after the initial interaction. The same cultures were photographed before their preparation for measuring thymidine incorporation. Figure 2 shows that few parasites (arrows) were present in E5C2, 72 hours after the initial interaction. In E9C5 and E8C6 cultures, however, a large number of tachyzoites was observed. 
ODC Activity
Because high tachyzoite growth was found in cultures from old embryos, whereas in vivo polyamines are known to be at low concentrations and with low activity levels of ODC, we evaluated ODC activity in our experimental model (dense, stationary cultures) as a function of their developmental stage. In agreement with a high proliferation rate of T. gondii in more developed cultures, ODC activity was found to be approximately three times higher in E8C6 and E9C5 than in E5C2 (Fig. 3)
ODC Expression in Chick Retina and Dense, Stationary Cultures
ODC immunoreactivity in freshly dissected retinas revealed that, at E8 (Fig. 4C) , almost all cells in the tissue were labeled for ODC, in agreement with the high activity of this enzyme at this developmental stage 49 At E14 (Fig. 4B) and in posthatch animals (Fig. 4A) , when ODC activity is low, a more restricted immunoreactivity was observed, with ganglion cells and cells in the inner nuclear layers more intensely labeled. The immunocytochemical analysis of dense, stationary cultures revealed that, at E5C2 (Fig. 4F) , clusters of immunoreactive cells were observed within a population of dispersed unlabeled cells, whereas at E8C6 (Fig. 4E) and E9C5 (Fig. 4D) almost all cells were positive for ODC. 
ADC Activity
An alternative pathway for polyamine synthesis, mainly in bacteria and protists, involves the conversion of arginine into agmatine by ADC. Agmatinase then catalyzes the conversion of agmatine directly to putrescine. The activity of ADC has never been studied in the retinal tissue and could be responsible for part of putrescine synthesis in the developing retina. The pattern of ADC activity in the developing intact tissue followed that observed by de Mello et al. 49 and Yamasaki et al. 53 for ODC in chick and rat retinas, being high in retinas from animals at the early stages of development and declining as a function of retinal differentiation (Fig. 5) . In dense, stationary cultures, as opposed to the intact tissue, the developmental pattern of ADC followed the same profile as that of ODC observed in this model. High activity levels were found in cultures from older embryos (E8C6 and E9C5, Fig. 6 ). 
The data described so far indicate that, in contrast to retinas differentiating in ovo, dissociated cultured cells display a delay in the dynamics of polyamine synthesis and seem to point to a relationship between high levels of putrescine synthesis and the efficiency of T. gondii proliferation in the embryonic avian retina. 
Parasite Growth in Cultured Retinal Explants
As shown herein for ADC and in previous publications for ODC, the activities of these enzymes are high in retinas from young embryos, declining with the differentiation of the tissue. Therefore, one would expect that, in cultured retina explant from young embryos, the growth of T. gondii would be more efficient than in explants prepared from more differentiated retinas. Figure 7 shows that, as expected, retinas obtained from embryos at E5 and cultured for 2 days allowed a proliferation of T. gondii that was four to five times higher than in E8C6 and E9C5. 
Influence of Inhibitors of Polyamine Synthesis
The possibility that T. gondii proliferation might depend on the production of polyamines by host cells was investigated by inhibiting ODC and/or ADC activities in cultured retina cells before infection. 
When dense, stationary cultures from 9-day-old chick embryos, were incubated with 10 mM α-methyl, ornithine 24 hours before and after cell–parasite interaction, the growth of parasites was reduced by approximately 38%. Moreover, the incubation of cells with either 10 mM α-difluoromethylornithine or 10 mM α-difluoromethylarginine for 48 hours before and after the interaction, resulted in reductions of parasite proliferation of approximately 40% and 20%, respectively (Fig. 8)
T. gondii Infection in Retina Cell Aggregate Cultures
Preliminary data on aggregate cultures showed that even with a retinal cell-tachyzoite ratio 100 times lower than that in dense, stationary culture interactions, parasites within retinal cells were always observed (Fig. 9) . Both neuronal and glial cells were infected. In most thin sections analyzed, E5C4 aggregates displayed a higher number of tachyzoites than did E9C4 aggregates (Fig. 9) . Although neurons were also infected by T. gondii, in all sections analyzed it seemed that most parasite cells were within glial-like structures. However, an appropriate quantitative study is needed to confirm this observation. 
Discussion
It has been suggested that cells from several brain regions are differently susceptible to T. gondii infection, depending on the developmental stage of the tissue (e.g., fetal versus neonatal). 54 The fact that substantial changes in the microenvironment of the CNS occur during development and that acquired and congenital retinochoroiditis are also associated with the developmental stages of infected individuals indicates that metabolic characteristics of the embryonic retinal tissue may be relevant to the susceptibility of this tissue to toxoplasmosis. The present study of different culture models of the avian embryonic retina showed that the efficacy of T. gondii proliferation was dependent on the developmental stage of the tissue and seemed to be associated, at least in part, with the polyamine content of the retina. 
Parasite protozoa usually need substrates from the host microenvironment to survive. Trypanosoma cruzi for instance is unable to synthesize putrescine, 55 56 57 58 and consequently its growth depends on polyamines from host cell. 55 The dependence on host polyamines has also been shown for T. gondii in a recent publication by Seabra et al. 48 They have shown that the enzymes responsible for the biosynthesis of putrescine, ODC and ADC, are absent in this protozoan. Moreover, they have shown that T. gondii is capable of taking up putrescine from the environment through a transporter system that displays a very low K m (0.9 μM) for the polyamine precursor putrescine. These data, added to the fact that ODC and ADC activities and putrescine levels are higher in retinas from young embryos than in those from older animals 49 and that congenital infection is associated with a higher risk of ocular involvement than postnatally acquired infection, 11 12 indicate that the metabolism of polyamines may be an important factor influencing T. gondii proliferation in this tissue. 
Contrary to our initial expectations, tachyzoite proliferation was high in cultures derived from older embryos. This was explained by the finding of high ADC and ODC activity levels in stationary cultures prepared from old embryos, as opposed to that observed in the normal tissue and in explant cultures. 49 This discrepancy is not restricted to ODC and ADC. Other parameters, such as dopamine-mediated cAMP accumulation 59 and glutamate decarboxylase activity are also altered when intact retinas are dissociated for culture seeding. 60 The reason for that seems to be related to the disruption of cell interactions during culture preparation. 61  
The partial reduction of tachyzoites proliferation in retina cultures previously treated with inhibitors of putrescine synthesis, strongly suggest that T. gondii requires polyamines of the infected tissue to grow. The fact that inhibition of putrescine synthesis did not completely arrest parasite growth may be due to the possibility that, as in mammalian host cells, 62 63 multiple routes of polyamine synthesis exist. Moreover, because T. gondii displays a putrescine transport system with a K m for this amine of the order of 0.9 μM, even low levels of putrescine concentration may be sufficient to support tachyzoite proliferation. In macrophages, for instance, the estimated constitutive intracellular concentration of putrescine is on the order of 100 μM, 48 58 sufficient to allow the proper multiplication of tachyzoites. Thus, if in susceptible retinal cells the concentration of putrescine were over the range of the K m of its transporter, even after a 48-hour inhibition of ODC and ADC activities, a residual growth of the parasite would be ascertained. 
The presence of ADC activity in both intact and cultured retinas suggests that this tissue may also accumulate agmatine. Besides serving as an alternative pathway for polyamine synthesis, agmatine may play an additional role in facilitating the proliferation of T. gondii. This compound is a potent inhibitor of nitric oxide synthase 64 65 and it has been reported that nitric oxide is an important suppressor of T. gondii development. 66  
That the retinal tissue is formed by almost 100 cell subtypes raises the possibility that toxoplasma proliferation or the stage conversion to the latent form (bradyzoite) is facilitated in some cells but not in others. Cellular features, like the presence of agmatine and perhaps γ-aminobutyric acid (GABA), which can also be synthesized from putrescine, 53 should be a matter of further studies concerning the toxoplasma–retinal tissue relationship. 
Aggregate cultures 67 68 69 of retina cells have been used by many investigators to approach the definition of many molecular phenotypes in vitro. Also, this culture system was used to study mechanisms of synaptogenesis, sorting out of retinal cells that normally occur during development and retina histogenesis. 60 61 67 68 69 70 71 72 73 74 75 76 77 78 79 80 In most cases, these biological phenomena that take place in the retina during development are also found in aggregates differentiating in vitro. 60 72 73 75 76 78 80 This culture system has never been used to access protozoan parasite interaction and multiplication in host cells. The data reported herein show that at a very low tachyzoite-to-retinal cell ratio, mixed together just after retina dissociation, an intense infection of the aggregates occurs in a short period of coculture. We believe that the simultaneous seeding of T. gondii tachyzoites and developing retinal cells, followed by histogenesis in vitro, may mimic the conditions of congenital infection in situ. The presence of infected cells within the retinal tissue during development is a physiopathological characteristic of the infected tissue and of the disease that follows. 
The aggregate cell culture system could also constitute an important tool for the study of the phenomenon of stage conversion in toxoplasmic retinochoroiditis, a key event in this recurrent eye affection. 
 
Figure 1.
 
T. gondii proliferation (mean ± SEM) in monolayer, dense, stationary cultures of embryonic retina cells. (A) Seventy-two hours after the initial interaction, T. gondii showed higher proliferation rates in cultures originating from older embryos (E8C6, E9C5). P < 0.001 when E8C6 or E9C5 cultures were compared with E5C2 (n = 8). (B) No difference in [3H]-thymidine incorporation was observed in infected cultures originating from the three different ages when compared with noninfected control cells 24 hours after initial interaction. (C) Forty-eight hours after initial interaction, an increase in [3H]-thymidine incorporation was observed in the three cultures used, but no statistically significant difference was observed (n = 8, P > 0.05).
Figure 1.
 
T. gondii proliferation (mean ± SEM) in monolayer, dense, stationary cultures of embryonic retina cells. (A) Seventy-two hours after the initial interaction, T. gondii showed higher proliferation rates in cultures originating from older embryos (E8C6, E9C5). P < 0.001 when E8C6 or E9C5 cultures were compared with E5C2 (n = 8). (B) No difference in [3H]-thymidine incorporation was observed in infected cultures originating from the three different ages when compared with noninfected control cells 24 hours after initial interaction. (C) Forty-eight hours after initial interaction, an increase in [3H]-thymidine incorporation was observed in the three cultures used, but no statistically significant difference was observed (n = 8, P > 0.05).
Figure 2.
 
Monolayer cultured embryonic retinal cells 72 hours after the initial interaction with T. gondii tachyzoites. (A, B) Cultures prepared with retinas from 5-day-old embryos maintained for 2 days in culture (E5C2); (C, D) E8C6; and (E, F) E9C5. Control noninfected cultures (A, C, E). Infected cultures (B, D, F). Arrows: tachyzoites.
Figure 2.
 
Monolayer cultured embryonic retinal cells 72 hours after the initial interaction with T. gondii tachyzoites. (A, B) Cultures prepared with retinas from 5-day-old embryos maintained for 2 days in culture (E5C2); (C, D) E8C6; and (E, F) E9C5. Control noninfected cultures (A, C, E). Infected cultures (B, D, F). Arrows: tachyzoites.
Figure 3.
 
ODC activity (mean ± SEM) in monolayer, stationary cultures of embryonic retinal cells. High activity levels were observed in those cultures originating from older embryos when compared with those from younger ones (n = 3; P < 0,05).
Figure 3.
 
ODC activity (mean ± SEM) in monolayer, stationary cultures of embryonic retinal cells. High activity levels were observed in those cultures originating from older embryos when compared with those from younger ones (n = 3; P < 0,05).
Figure 4.
 
ODC immunoreactivity in radial sections of retinas from (A) adult and (B) 14- and (C) 8-day-old chick embryos and in monolayer stationary retinal cell cultures originated from chick embryo (E9C5, D; E8C6, E; and E5C2, F). (A, B, arrows) Examples of labeled cells in both GCL and INL. (D, E, Image not available ) Labeled cells and (F) clusters in monolayer cultures. Right panels: control cultures. INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; NL, neuroblastic layer.
Figure 4.
 
ODC immunoreactivity in radial sections of retinas from (A) adult and (B) 14- and (C) 8-day-old chick embryos and in monolayer stationary retinal cell cultures originated from chick embryo (E9C5, D; E8C6, E; and E5C2, F). (A, B, arrows) Examples of labeled cells in both GCL and INL. (D, E, Image not available ) Labeled cells and (F) clusters in monolayer cultures. Right panels: control cultures. INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; NL, neuroblastic layer.
Figure 5.
 
ADC activity (mean ± SEM) in intact retinal tissue. Retinas from young embryos showed higher ADC activity when compared with those from older ones. ADC activity was statistically different from all other stages studied (E7 vs. E9 P < 0.05; E7 vs. E14 P < 0.001; E9 vs. E14; P < 0.05; n = 3).
Figure 5.
 
ADC activity (mean ± SEM) in intact retinal tissue. Retinas from young embryos showed higher ADC activity when compared with those from older ones. ADC activity was statistically different from all other stages studied (E7 vs. E9 P < 0.05; E7 vs. E14 P < 0.001; E9 vs. E14; P < 0.05; n = 3).
Figure 6.
 
ADC activity (mean ± SEM) in monolayer cultures of embryonic retina. High activity levels were observed in those cultures originated from old embryos (E8C6 and E9C5). P < 0.05 when compared with E5C2.
Figure 6.
 
ADC activity (mean ± SEM) in monolayer cultures of embryonic retina. High activity levels were observed in those cultures originated from old embryos (E8C6 and E9C5). P < 0.05 when compared with E5C2.
Figure 7.
 
T. gondii proliferation (mean ± SEM) in explant cultures of embryonic retina cells 48 hours after initial interaction. Higher proliferation rates were observed in cultures originated from younger than older embryos (E5C2). (E8C6 vs. E5C2 P < 0.001; E9C5 vs. E5C2 P < 0.01; n = 3.)
Figure 7.
 
T. gondii proliferation (mean ± SEM) in explant cultures of embryonic retina cells 48 hours after initial interaction. Higher proliferation rates were observed in cultures originated from younger than older embryos (E5C2). (E8C6 vs. E5C2 P < 0.001; E9C5 vs. E5C2 P < 0.01; n = 3.)
Figure 8.
 
Effect of inhibitors of ODC and ADC activities (mean ± SEM) in the parasite growth in monolayer stationary retinal cell cultures originating from 9-day-old embryos (E9C5). [3H]-Thymidine incorporation (mean ± SEM) was measured 72 hours after the initial interaction. The incubation of 10 mM methyl ornithine, 24 hours before and after interaction, reduced parasite growth by approximately 38% (P < 0.001; n = 8). The use of 10 mM difluoromethylornithine (DFMO) and 10 mM difluoromethylarginine (DFMA), 48 hours before and after interaction, resulted in a 40% (P < 0.01; n = 4) and 20% (P < 0.05; n = 3) reduction of parasite proliferation, respectively.
Figure 8.
 
Effect of inhibitors of ODC and ADC activities (mean ± SEM) in the parasite growth in monolayer stationary retinal cell cultures originating from 9-day-old embryos (E9C5). [3H]-Thymidine incorporation (mean ± SEM) was measured 72 hours after the initial interaction. The incubation of 10 mM methyl ornithine, 24 hours before and after interaction, reduced parasite growth by approximately 38% (P < 0.001; n = 8). The use of 10 mM difluoromethylornithine (DFMO) and 10 mM difluoromethylarginine (DFMA), 48 hours before and after interaction, resulted in a 40% (P < 0.01; n = 4) and 20% (P < 0.05; n = 3) reduction of parasite proliferation, respectively.
Figure 9.
 
Transmission electron micrograph of thin sections of cultured aggregated retina cells infected with T. gondii (RH strain). (A) Several parasites (Tx) are observed in a characteristic parasitophorous vacuole. (B) Parasite undergoing typical process of division (arrow). (C) Single parasite. N, cell nucleus.
Figure 9.
 
Transmission electron micrograph of thin sections of cultured aggregated retina cells infected with T. gondii (RH strain). (A) Several parasites (Tx) are observed in a characteristic parasitophorous vacuole. (B) Parasite undergoing typical process of division (arrow). (C) Single parasite. N, cell nucleus.
The authors thank Antonio Bosco, Eliandro Joaci de Lima, Marlene Cazuza, and Rosilane Taveira da Silva for excellent technical assistance; Ana Claudia Rozenfeld, Lucas Dantas Leite, Leandro Lengruber Soares, Sergio H. Seabra, and Solange for help in obtaining T. gondii tachyzoites; and Silvina Cejas, Juliana Kalaf, and Eliezer Israel Benchimol (Department of Ophthalmology, Federal University of Rio de Janeiro) for helpful discussions in the initial phases of the study. 
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Figure 1.
 
T. gondii proliferation (mean ± SEM) in monolayer, dense, stationary cultures of embryonic retina cells. (A) Seventy-two hours after the initial interaction, T. gondii showed higher proliferation rates in cultures originating from older embryos (E8C6, E9C5). P < 0.001 when E8C6 or E9C5 cultures were compared with E5C2 (n = 8). (B) No difference in [3H]-thymidine incorporation was observed in infected cultures originating from the three different ages when compared with noninfected control cells 24 hours after initial interaction. (C) Forty-eight hours after initial interaction, an increase in [3H]-thymidine incorporation was observed in the three cultures used, but no statistically significant difference was observed (n = 8, P > 0.05).
Figure 1.
 
T. gondii proliferation (mean ± SEM) in monolayer, dense, stationary cultures of embryonic retina cells. (A) Seventy-two hours after the initial interaction, T. gondii showed higher proliferation rates in cultures originating from older embryos (E8C6, E9C5). P < 0.001 when E8C6 or E9C5 cultures were compared with E5C2 (n = 8). (B) No difference in [3H]-thymidine incorporation was observed in infected cultures originating from the three different ages when compared with noninfected control cells 24 hours after initial interaction. (C) Forty-eight hours after initial interaction, an increase in [3H]-thymidine incorporation was observed in the three cultures used, but no statistically significant difference was observed (n = 8, P > 0.05).
Figure 2.
 
Monolayer cultured embryonic retinal cells 72 hours after the initial interaction with T. gondii tachyzoites. (A, B) Cultures prepared with retinas from 5-day-old embryos maintained for 2 days in culture (E5C2); (C, D) E8C6; and (E, F) E9C5. Control noninfected cultures (A, C, E). Infected cultures (B, D, F). Arrows: tachyzoites.
Figure 2.
 
Monolayer cultured embryonic retinal cells 72 hours after the initial interaction with T. gondii tachyzoites. (A, B) Cultures prepared with retinas from 5-day-old embryos maintained for 2 days in culture (E5C2); (C, D) E8C6; and (E, F) E9C5. Control noninfected cultures (A, C, E). Infected cultures (B, D, F). Arrows: tachyzoites.
Figure 3.
 
ODC activity (mean ± SEM) in monolayer, stationary cultures of embryonic retinal cells. High activity levels were observed in those cultures originating from older embryos when compared with those from younger ones (n = 3; P < 0,05).
Figure 3.
 
ODC activity (mean ± SEM) in monolayer, stationary cultures of embryonic retinal cells. High activity levels were observed in those cultures originating from older embryos when compared with those from younger ones (n = 3; P < 0,05).
Figure 4.
 
ODC immunoreactivity in radial sections of retinas from (A) adult and (B) 14- and (C) 8-day-old chick embryos and in monolayer stationary retinal cell cultures originated from chick embryo (E9C5, D; E8C6, E; and E5C2, F). (A, B, arrows) Examples of labeled cells in both GCL and INL. (D, E, Image not available ) Labeled cells and (F) clusters in monolayer cultures. Right panels: control cultures. INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; NL, neuroblastic layer.
Figure 4.
 
ODC immunoreactivity in radial sections of retinas from (A) adult and (B) 14- and (C) 8-day-old chick embryos and in monolayer stationary retinal cell cultures originated from chick embryo (E9C5, D; E8C6, E; and E5C2, F). (A, B, arrows) Examples of labeled cells in both GCL and INL. (D, E, Image not available ) Labeled cells and (F) clusters in monolayer cultures. Right panels: control cultures. INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; NL, neuroblastic layer.
Figure 5.
 
ADC activity (mean ± SEM) in intact retinal tissue. Retinas from young embryos showed higher ADC activity when compared with those from older ones. ADC activity was statistically different from all other stages studied (E7 vs. E9 P < 0.05; E7 vs. E14 P < 0.001; E9 vs. E14; P < 0.05; n = 3).
Figure 5.
 
ADC activity (mean ± SEM) in intact retinal tissue. Retinas from young embryos showed higher ADC activity when compared with those from older ones. ADC activity was statistically different from all other stages studied (E7 vs. E9 P < 0.05; E7 vs. E14 P < 0.001; E9 vs. E14; P < 0.05; n = 3).
Figure 6.
 
ADC activity (mean ± SEM) in monolayer cultures of embryonic retina. High activity levels were observed in those cultures originated from old embryos (E8C6 and E9C5). P < 0.05 when compared with E5C2.
Figure 6.
 
ADC activity (mean ± SEM) in monolayer cultures of embryonic retina. High activity levels were observed in those cultures originated from old embryos (E8C6 and E9C5). P < 0.05 when compared with E5C2.
Figure 7.
 
T. gondii proliferation (mean ± SEM) in explant cultures of embryonic retina cells 48 hours after initial interaction. Higher proliferation rates were observed in cultures originated from younger than older embryos (E5C2). (E8C6 vs. E5C2 P < 0.001; E9C5 vs. E5C2 P < 0.01; n = 3.)
Figure 7.
 
T. gondii proliferation (mean ± SEM) in explant cultures of embryonic retina cells 48 hours after initial interaction. Higher proliferation rates were observed in cultures originated from younger than older embryos (E5C2). (E8C6 vs. E5C2 P < 0.001; E9C5 vs. E5C2 P < 0.01; n = 3.)
Figure 8.
 
Effect of inhibitors of ODC and ADC activities (mean ± SEM) in the parasite growth in monolayer stationary retinal cell cultures originating from 9-day-old embryos (E9C5). [3H]-Thymidine incorporation (mean ± SEM) was measured 72 hours after the initial interaction. The incubation of 10 mM methyl ornithine, 24 hours before and after interaction, reduced parasite growth by approximately 38% (P < 0.001; n = 8). The use of 10 mM difluoromethylornithine (DFMO) and 10 mM difluoromethylarginine (DFMA), 48 hours before and after interaction, resulted in a 40% (P < 0.01; n = 4) and 20% (P < 0.05; n = 3) reduction of parasite proliferation, respectively.
Figure 8.
 
Effect of inhibitors of ODC and ADC activities (mean ± SEM) in the parasite growth in monolayer stationary retinal cell cultures originating from 9-day-old embryos (E9C5). [3H]-Thymidine incorporation (mean ± SEM) was measured 72 hours after the initial interaction. The incubation of 10 mM methyl ornithine, 24 hours before and after interaction, reduced parasite growth by approximately 38% (P < 0.001; n = 8). The use of 10 mM difluoromethylornithine (DFMO) and 10 mM difluoromethylarginine (DFMA), 48 hours before and after interaction, resulted in a 40% (P < 0.01; n = 4) and 20% (P < 0.05; n = 3) reduction of parasite proliferation, respectively.
Figure 9.
 
Transmission electron micrograph of thin sections of cultured aggregated retina cells infected with T. gondii (RH strain). (A) Several parasites (Tx) are observed in a characteristic parasitophorous vacuole. (B) Parasite undergoing typical process of division (arrow). (C) Single parasite. N, cell nucleus.
Figure 9.
 
Transmission electron micrograph of thin sections of cultured aggregated retina cells infected with T. gondii (RH strain). (A) Several parasites (Tx) are observed in a characteristic parasitophorous vacuole. (B) Parasite undergoing typical process of division (arrow). (C) Single parasite. N, cell nucleus.
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